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| United States Patent |
6,265,375
|
|
Gilon
, et al.
|
July 24, 2001
|
Conformationally constrained backbone cyclized peptide analogs
Abstract
Novel backbone cyclized peptide analogs are formed by means of bridging
groups attached via the alpha nitrogens of amino acid derivatives to
provide novel non-peptidic linkages. Novel building units disclosed are
N.sup..alpha. (.omega.-functionalized) amino acids constructed to include
a spacer and a terminal functional group. One or more of these
N.sup..alpha. (.omega.-functionalized) amino acids are incorporated into a
peptide sequence, preferably during solid phase peptide synthesis. The
reactive terminal functional groups are protected by specific protecting
groups that can be selectively removed to effect either
backbone-to-backbone or backbone-to-side chain cyclizations. The invention
is specifically exemplified by backbone cyclized bradykinin antagonists
having biological activity. Further embodiments of the invention are
somatostatin analogs having one or two ring structures involving backbone
cyclization.
| Inventors:
|
Gilon; Chaim (Jerusalem, IL);
Eren; Doron (Rehovot, IL);
Zeltser; Irina (Jerusalem, IL);
Seri-Levy; Alon (Jerusalem, IL);
Gitan; Gal (Jerusalem, IL);
Muller; Dan (Jerusalem, IL)
|
| Assignee:
|
Yissum Research Development Co. of the Hebrew University (Jerusalem, IL);
Peptor Limited (Rehovot, IL)
|
| Appl. No.:
|
120237 |
| Filed:
|
July 22, 1998 |
Foreign Application Priority Data
| Current U.S. Class: |
514/9; 514/10; 514/15; 514/16; 530/311; 530/317; 530/318 |
| Intern'l Class: |
A61K 038/08; A61K 038/12; C07K 007/64 |
| Field of Search: |
530/311,317,328
514/9,10,11,15,16
|
References Cited
U.S. Patent Documents
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| 4235886 | Nov., 1980 | Freidinger et al. | 424/177.
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| WO 93/01206 | Jan., 1993 | WO.
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| WO 94/11393 | May., 1994 | WO.
| |
Other References
Bell et al., "Molecular biology of somatostatin receptors", TINS, vol. 16,
No. 1, 34-38.
Brazeau et al. 1973, "Hypothalamic Polypeptide That Inhibits the Secretion
of Immunoreative Pituitary Growth Hormone", Science, vol. 179, pp. 77-79.
Buscail et al., 1995, "Inhibition of cell proliferation by the somatostatin
analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and
SSTR5 through different mechanisms", Proc. Natl. Acad. Sci. USA
92:1580-1584.
Byk et al., 1992, "Building units for N-backbone cyclic peptides. 1.
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Charpentier et al., 1989, "Synthesis and Binding Affinities of Cyclic and
Related Linear Analogues of CCK.sub.8 Selective for Central Receptors", J.
Med. Chem., pp. 1184-1190.
Giannis et al., 1993, "Peptidomimetics for Receptor Ligands--Discovery,
Development, and Medical Perspectives", Angew. Chem. Int. Ed. Engl.
32:1244-1267.
Gilon et al., 1991, "Backbone Cyclization: A New Method for Conferring
Conformational Constraints on Peptides", Biopolymers 31:745-750.
Gilon et al., 1992, "SAR studies of cycloseptide: Effects of cyclization
and charge at position 6", Chem. Biol. Proc Am Pept Symp 1th. pp. 476-477.
Greiner et al., 1994, "Synthesis of New Backbone-Cyclized Bradykinin
Analogs", Proc. Eur. Pept. Symp., 23rd, Meeting Date 1994, 289-290.
Hruby et al., 1990, "Emerging approaches in the molecfular design of
receptor-selective peptide ligands: conformational, topographical and
dynamic considerations", Biochem J. 268:249-262.
Krstenansky et al., 1994, "Cyclic hexapeptide antagonists of the bradykinin
B.sub.2 receptor: Receptor binding and solution backbone conformation",
Letters in Peptide Science 1:229-234.
Lamberts, 1988, "The Role of Somatostatin in the Regulation of Anterior
Pituitary Hormone Secretion and the Use of Its Analogs in the Treatment of
Human Pituitary Tumors", Endocrine Reviews vol. 9, No. 4, pp. 417-436.
Lamberts et al., 1990, "Somatostatin-receptor imaging in the localization
of endocrine tumors", New England J. Med. 323:1246-1249.
Lymangrover et al., 1993, "Varying the Duration of A23187 Administration
Alters its Effect on Adrenal Steroidogenesis", Life Sciences 34:371-377.
Mosberg et al., 1983, "Bis-penicillamine enkephalins possess highly
improved specificity toward .delta. opioid receptors", Biochemistry
80:5871-5874.
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somatostatin secretion into the hypophysial-portal circulation of the
rat", Science 230:461-463.
Raynor et al., 1993, "Cloned somatostatin receptors: Identification of
subtype-selective peptides and demonstration of high affinity binding of
linear peptides", Mol. Pharmacol. 43:838-844.
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Endocrine Reviews 16:427-442.
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disease," TIPS 16:110-115.
Rizo et al., 1992, "Constrained Peptides: Models of Bioactive Peptides and
Protein Substructures", Annu. Rev. Biochem. 61:387-418.
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selective for central receptors", Int. J. Peptide Protein Res. 35:441-451.
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localized to sensory neurons, and antagonists have analgesic actions",
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somatostatin", Life Sciences 34:1371-1378.
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Current Opinion in Structural Biol. 3:580-584.
|
Primary Examiner: Low; Christopher S. F.
Assistant Examiner: Gupta; Anish
Attorney, Agent or Firm: Pennie & Edmonds LLP
Parent Case Text
This is a continuation of application Ser. No. 08/488,159, filed Jun. 7,
1995 now U.S. Pat. No. 5,811,392.
Claims
What is claimed is:
1. A backbone cyclized peptide analog having the general Formula (I):
##STR47##
wherein: a and b each independently designates an integer from 1 to 8 or
zero; d, e, and f each independently designates an integer from 1 to 10;
(AA) designates an amino acid residue wherein the amino acid residues in
each chain may be the same or different; E represents a hydroxyl group, a
carboxyl protecting group or an amino group, or CO--E can be reduced to
CH.sub.2 --OH; each of R, R', R", and R'" is independently hydrogen or an
amino acid side-chain optionally bound with a specific protecting group;
and the lines designate a bridging group of the Formula:
(i) --X--M--Y--W--Z--; or (ii) --X--M--Z--
wherein: one line may be absent; M and W are independently selected from
the group consisting of disulfide, amide, thioether, thioester, imine,
ether, and alkene; and X, Y and Z are each independently selected from the
group consisting of alkylene, substituted alkylene, arylene, homo- or
hetero-cycloalkylene and substituted cycloalkylene.
2. The backbone cyclized peptide analog of claim 1 wherein
--X--M--Y--W--Z-- is:
--(CH.sub.2).sub.x --M--(CH.sub.2).sub.y --W--(CH.sub.2).sub.z --
wherein M and W are independently selected from the group consisting of
disulfide, amide, thioether, thioester, imine, ether, and alkene; x and z
each independently designates an integer of from 1 to 10, and y is zero or
an integer of from 1 to 8, with the proviso that if y is zero, W is
absent.
3. The backbone cyclized peptide analog of claim 1 wherein the group CO--E
is CH.sub.2 OH.
4. The backbone cyclized peptide analog of claim 1 wherein R is CH.sub.3
--, (CH.sub.3).sub.2 CH--, (CH.sub.3).sub.2 CHCH.sub.2 --, CH.sub.3
CH.sub.2 CH(CH.sub.3)--, CH.sub.3 S(CH.sub.2).sub.2 --, HOCH.sub.2 --,
CH.sub.3 CH(OH)--, HSCH.sub.2 --, NH.sub.2 C(.dbd.O)CH.sub.2 --, NH.sub.2
C(.dbd.O)(CH.sub.2).sub.2 --, NH.sub.2 (CH.sub.2).sub.3 --,
HOC(.dbd.O)CH.sub.2 --, HOC(.dbd.O)(CH.sub.2).sub.2 --, NH.sub.2
(CH.sub.2).sub.4 --, C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --,
HO-phenyl-CH.sub.2 --, benzyl, methylindole, or methylimidazole.
5. The backbone cyclized peptide analog of claim 1 wherein R' is CH.sub.3,
(CH.sub.3).sub.2 CH--, (CH.sub.3).sub.2 CHCH.sub.2 --, CH.sub.3 CH.sub.2
CH(CH.sub.3)--, CH.sub.3 S(CH.sub.2).sub.2 --, HOCH.sub.2 --, CH.sub.3
CH(OH)--, HSCH.sub.2 --, NH.sub.2 C(.dbd.O)CH.sub.2 --, NH.sub.2
C(.dbd.O)(CH.sub.2).sub.2 --, C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --,
HOC(.dbd.O)CH.sub.2 --, HOC(.dbd.)(CH.sub.2 --, NH.sub.2 (CH.sub.2).sub.4
--, C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --, HO-phenyl-CH.sub.2 --,
benzyl, methylindole, or methylimidazole.
6. A backbone cyclized peptide analog having the general Formula (XIVa):
##STR48##
wherein m and n are 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ; R.sup.5 is
absent or is Gly, (D)- or (L)-Ala, Phe, Nal, and .beta.-Asp(Ind); R.sup.6
and R.sup.11 are independently Gly or (D)- or (L)-Phe; R.sup.7 is Phe or
Tyr; R.sup.10 to is absent or is Gly, Abu, Thr or Val; R.sup.12 is absent
or is Thr or Nal; and Y.sup.2 is selected from the group consisting of
amide, disulfide, thioether, imine, ether, and alkene.
7. A backbone cyclized peptide analog having the general Formula (XIVb):
##STR49##
wherein m and n are 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ; R.sup.6 and
R.sup.11 are independently Gly or (D)- or (L)-Phe; R.sup.7 is Phe or Tyr;
R.sup.10 is absent or is Gly, Abu, Thr or Val; and Y.sup.2 is selected
from the group consisting of amide, disulfide, thioether, imine, ether,
and alkene.
8. A backbone cyclized peptide analog having the general Formula (XVa):
##STR50##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R.sup.6
is (D) or (L)-Phe; R.sup.10 is absent or is Gly, Abu or Thr; and R.sup.11
is (D)- or (L)-Phe; R.sup.12 is absent or is Thr or Nal, and Y.sup.1 is
selected from the group consisting of amide, disulfide, thioether, imine,
ether, and alkene.
9. A backbone cyclized peptide analog having the general Formula (XVb):
##STR51##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.6 is (D) or (L)-Phe; R.sup.10 is absent or is Gly, Abu or Thr; and
R.sup.11 is (D)- or (L)-Phe; R.sup.12 is absent or is Thr or Nal, and
Y.sup.1 is selected from the group consisting of amide, disulfide,
thioether, imine, ether, and alkene.
10. A backbone cyclized peptide analog having the general Formula (XVIa):
##STR52##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R.sup.6
is (D) or (L)-Phe; R.sup.10 is absent or is Gly, Abu or Thr; and R.sup.11
is (D)- or (L)-Phe; R.sup.12 is absent or is Thr or Nal, and Y.sup.1 is
selected from the group consisting of amide, disulfide, thioether, imine,
ether, and alkene.
11. A backbone cyclized peptide analog having the general Formula (XVIb):
##STR53##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.6 is (D) or (L)-Phe; R.sup.10 is absent or is Gly, Abu or Thr; and
R.sup.11 is (D)- or (L)-Phe; R.sup.12 is absent or is Thr or Nal, and
Y.sup.1 is selected from the group consisting of amide, disulfide,
thioether, imine, ether, and alkene.
12. A backbone cyclized peptide analog having the general Formula (XVIc):
##STR54##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R.sup.6
is (D) or (L)-Phe; and R.sup.10 is absent or is Gly, Abu or Thr; R.sup.12
is absent or is Thr or Nal, and Y.sup.1 is selected from the group
consisting of amide, disulfide, thioether, imine, ether, and alkene.
13. The backbone cyclized peptide analog of claim 1 having the general
Formula (XIXa):
##STR55##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R.sup.10
is absent or is Gly, Abu or Thr; R.sup.12 is absent or is Thr or Nal; and
Y.sup.1 is selected from the group consisting of amide, disulfide,
thioether, imine, ether, and alkene.
14. The backbone cyclized peptide analog of claim 1 having the general
Formula (XIXb):
##STR56##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
and Y.sup.1 is selected from the group consisting of amide, disulfide,
thioether, imine, ether, and alkene.
15. The backbone cyclized peptide analog of claim 1 having the general
Formula (XXa):
##STR57##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R.sup.10
is absent or is Gly, Abu or Thr; R.sup.12 is absent or is Thr or Nal; and
Y.sup.1 is selected from the group consisting of amide, disulfide,
thioether, imine, ether, and alkene.
16. The backbone cyclized peptide analog of claim 1 having the general
Formula (XXb):
##STR58##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.10 is absent or is Gly, Abu or Thr; R.sup.12 is absent or is Thr or
Nal; and Y.sup.1 is selected from the group consisting of amide,
disulfide, thioether, imine, ether, and alkene.
17. A pharmaceutical composition comprising the backbone cyclized peptide
analog of claim 1 and a pharmaceutically acceptable carrier or diluent.
Description
FIELD OF THE INVENTION
The present invention relates to conformationally constrained N.sup..alpha.
backbone-cyclized peptide analogs cyclized via novel non-peptidic
linkages, to novel N.sup..alpha.,.omega.-functionalized amino acid
building units, to processes for the preparation of these backbone
cyclized peptides and building units, to methods for using these peptide
analogs and to pharmaceutical compositions containing same.
BACKGROUND OF THE INVENTION
Peptidomimetics
As a result of major advances in organic chemistry and in molecular
biology, many bioactive peptides can now be prepared in quantities
sufficient for pharmacological and clinical utilities. Thus in the last
few years new methods have been established for the treatment and therapy
of illnesses in which peptides have been implicated. However, the use of
peptides as drugs is limited by the following factors: a) their low
metabolic stability towards proteolysis in the gastrointestinal tract and
in serum; b) their poor absorption after oral ingestion, in particular due
to their relatively high molecular mass or the lack of specific transport
systems or both; c) their rapid excretion through the liver and kidneys;
and d) their undesired side effects in non-target organ systems, since
peptide receptors can be widely distributed in an organism.
Moreover, with few exceptions, native peptides of small to medium size
(less than 30-50 amino acids) exist unordered in dilute aqueous solution
in a multitude of conformations in dynamic equilibrium which may lead to
lack of receptor selectivity, metabolic susceptibilities and hamper
attempts to determine the biologically active conformation. If a peptide
has the biologically active conformation per se, i.e., receptor-bound
conformation, then an increased affinity toward the receptor is expected,
since the decrease in entropy on binding is less than that on the binding
of a flexible peptide. It is therefore important to strive for and develop
ordered, uniform and biologically active peptides.
In recent years, intensive efforts have been made to develop
peptidomimetics or peptide analogs that display more favorable
pharmacological properties than their prototype native peptides. The
native peptide itself, the pharmacological properties of which have been
optimized, generally serves as a lead for the development of these
peptidomimetics. However, a major problem in the development of such
agents is the discovery of the active region of a biologically active
peptide. For instance, frequently only a small number of amino acids
(usually four to eight) are responsible for the recognition of a peptide
ligand by a receptor. Once this biologically active site is determined a
lead structure for development of peptidomimetic can be optimized, for
example by molecular modeling programs.
As used herein, a "peptidomimetic" is a compound that, as a ligand of a
receptor, can imitate (agonist) or block (antagonist) the biological
effect of a peptide at the receptor level. The following factors should be
considered to achieve the best possible agonist peptidomimetic a)
metabolic stability, b) good bioavailability, c) high receptor affinity
and receptor selectivity, and d) minimal side effects.
From the pharmacological and medical viewpoint it is frequently desirable
to not only imitate the effect of the peptide at the receptor level
(agonism) but also to block the receptor when required (antagonism). The
same pharmacological considerations for designing an agonist
peptidomimetic mentioned above hold for designing peptide antagonists,
but, in addition, their development in the absence of lead structures is
more difficult. Even today it is not unequivocally clear which factors are
decisive for the agonistic effect and which are for the antagonistic
effect.
A generally applicable and successful method recently has been the
development of conformationally restricted peptidomimetics that imitate
the receptor-bound conformation of the endogenous peptide ligands as
closely as possible (Rizo and Gierasch, Ann. Rev. Biochem., 61:387, 1992).
Investigations of these types of analogs show them to have increased
resistance toward proteases, that is, an increase in metabolic stability,
as well as increased selectivity and thereby fewer side effects (Veber and
Friedinger, Trends Neurosci., p. 392, 1985).
Once these peptidomimetic compounds with rigid conformations are produced,
the most active structures are selected by studying the
conformation-activity relationships. Such conformational constraints can
involve short range (local) modifications of structure or long range
(global) conformational restraints (for review see Giannis and Kolter,
Angew. Chem. Int. Ed. Engl. 32:1244, 1993).
Conformationally Constrained Peptides
Bridging between two neighboring amino acids in a peptide leads to a local
conformational modification, the flexibility of which is limited in
comparison with that of regular dipeptides. Some possibilities for forming
such bridges include incorporation of lactams and piperazinones.
.gamma.-Lactams and .delta.-lactams have been designed to some extent as
"turn mimetics"; in several cases the incorporation of such structures
into peptides leads to biologically active compounds.
Global restrictions in the conformation of a peptide are possible by
limiting the flexibility of the peptide strand through cyclization (Hruby
et al., Biochem. J., 268:249, 1990). Not only does cyclization of
bioactive peptides improve their metabolic stability and receptor
selectivity, cyclization also imposes constraints that enhance
conformational homogeneity and facilitates conformational analysis. The
common modes of cyclization are the same found in naturally occurring
cyclic peptides. These include side chain to side chain cyclization or
side chain to end-group cyclization. For this purpose, amino acid side
chains that are not involved in receptor recognition are connected
together or to the peptide backbone. Another common cyclization is the
end-to-end cyclization.
Three representative examples are compounds wherein partial structures of
each peptide are made into rings by linking two penicillamine residues
with a disulfide bridge (Mosberg et al., P.N.A.S. US, 80:5871, 1983), by
formation of an amide bond between a lysine and an aspartate group
(Charpentier et al., J. Med. Chem. 32:1184, 1989), or by connecting two
lysine groups with a succinate unit (Rodriguez et al., Int. J. Pept.
Protein Res. 35:441, 1990). These structures have been disclosed in the
literature in the case of a cyclic enkephalin analog with selectivity for
the .delta.-opiate receptor (Mosberg et al., ibid.); or as agonists to the
cholecystokinin B receptor, found largely in the brain (Charpentier et
al., ibid., Rodriguez et al., ibid.).
The main limitations to these classical modes of cyclization are that they
require substitution of amino acid side chains in order to achieve
cyclization.
Another conceptual approach to the conformational constraint of peptides
was introduced by Gilon, et al., (Biopolymers, 31:745, 1991) who proposed
backbone to backbone cyclization of peptides. The theoretical advantages
of this strategy include the ability to effect cyclization via the carbons
or nitrogens of the peptide backbone without interfering with side chains
that may be crucial for interaction with the specific receptor of a given
peptide. While the concept was envisaged as being applicable to any linear
peptide of interest, in point of fact the limiting factor in the proposed
scheme was the availability of suitable building units that must be used
to replace the amino acids that are to be linked via bridging groups. The
actual reduction to practice of this concept of backbone cyclization was
prevented by the inability to devise any practical method of preparing
building units of amino acids other than glycine (Byk et al., J. Org.
Chem., 587:5687, 1992). While analogs of other amino acids were attempted
the synthetic method used was unsuccessful or of such low yield as to
preclude any general applicability.
In Gilon, EPO Application No. 564,739 A2; and J. Org. Chem., 57:5687, 1992,
two basic approaches to the synthesis of building units are described. The
first starts with the reaction of a diamine with a general .alpha. bromo
acid. Selective protection of the .omega. amine and further elaborations
of protecting groups provides a building unit, suitable for Boc chemistry
peptide synthesis. The second approach starts with selective protection of
a diamine and reaction of the product with chloroacetic acid to provide
the protected glycine derivative, suitable for Fmoc peptide synthesis.
Both examples deal with the reaction of a molecule of the general type
X--CH(R)--CO--OR' (wherein X represents a leaving group which, in the
examples given, is either Br or Cl) with an amine which replaces the X.
The amine bears an alkylidene chain which is terminated by another
functional group, amine in the examples described, which may or may not be
blocked by a protecting group.
In all cases the .alpha. nitrogen of the end product originates in the
molecule which becomes the bridging chain for subsequent cyclization. This
approach was chosen in order to take advantage of the higher
susceptibility to nucleophilic displacement of a leaving group next to a
carboxylic group.
In a molecule where R is different than hydrogen there is a high tendency
to eliminate HX under basic conditions. This side reaction reduces the
yield of Gilon's method to the point where it is impractical for
production of building units based on amino acids other than glycine. The
diamine nitrogen is primary while the product contains a secondary
nitrogen, which is a better nucleophile. So while the desired reaction may
be sluggish, and require the addition of catalysts, the product may be
contaminated with double alkylation products. There is no mention of
building units with end group chemistries other than nitrogen, so the only
cyclization schemes possible are backbone to side chain and backbone to C
terminus.
Applications of Conformationally Constrained Peptides
Conformationally constrained peptides find many pharmacological uses.
Somatostatin is a cyclic tetradecapeptide found both in the central
nervous system and in peripheral tissues. It was originally isolated from
mammalian hypothalamus and identified as an important inhibitor of growth
hormone secretion from the anterior pituitary. Its multiple biological
activities include inhibition of the secretion of glucagon and insulin
from the pancreas, regulation of most gut hormones and regulation of the
release of other neurotransmitters involved in motor activity and
cognitive processes throughout the central nervous system (for review see
Lamberts, Endocrine Rev., 9:427, 1988).
Natural somatostatin (also known as Somatotropin Release Inhibiting Factor,
SRIF) of the following structure:
H-Ala.sup.1 -Gly.sup.2 -Cys.sup.3 -Lys.sup.4 -Asn.sup.5 -Phe.sup.6
-Phe.sup.7 -Trp.sup.8 -Lys.sup.9 -Thr.sup.10 -Phe.sup.11 -Thr.sup.12
-Ser.sup.13 -Cys.sup.14 -OH
was first isolated by Guillemin and colleagues (Brazeau et al. Science,
179:78, 1973). In its natural form, it has limited use as a therapeutic
agent since it exhibits two undesirable properties: poor bioavailability
and short duration of action. For this reason, great efforts have been
made during the last two decades to find somatostatin analogs that will
have superiority in either potency, biostability, duration of action or
selectivity with regard to inhibition of the release of growth hormone,
insulin or glucagon.
Structure-activity relation studies, spectroscopic techniques such as
circular dichroism and nuclear magnetic resonance, and molecular modeling
approaches reveal the following: the conformation of the cyclic part of
natural somatostatin is most likely to be an antiparallel .beta.-sheet;
Phe.sup.6 and Phe.sup.11 play an important role in stabilizing the
pharmacophore conformation through hydrophobic interactions between the
two aromatic rings; the four amino acids Phe.sup.7 -Trp.sup.8 -Lys.sup.9
-Thr.sup.10 which are spread around the .beta.-turn in the antiparallel
.beta.-sheet are essential for the pharmacophore; and (D)Trp.sup.8 is
almost always preferable to (L)Trp.sup.8.
Nevertheless, a hexapeptide somatostatin analog containing these four amino
acids anchored by a disulfide bridge:
##STR1##
is almost inactive both in vitro and in vivo, although it has the advantage
of the covalent disulfide bridge which replaces the Phe.sup.6 -Phe.sup.11
hydrophobic interactions in natural somatostatin.
Four main approaches have been attempted in order to increase the activity
of this hexapeptide somatostatin analog. (1) Replacing the disulfide
bridge by a cyclization which encourages a cis-amide bond, or by
performing a second cyclization to the molecule yielding a bicyclic
analog. In both cases the resultant analog has a reduced number of
conformational degrees of freedom. (2) Replacing the original amino acids
in the sequence Phe.sup.7 -(D)Trp.sup.8 -Lys.sup.9 -Thr.sup.10 with more
potent amino acid analogs, such as replacing Phe.sup.7 with Tyr.sup.7 and
Thr.sup.10 with Val.sup.10. (3) Incorporating additional structural
elements from natural somatostatin with the intention that these new
elements will contribute to the interaction with the receptor. (4)
Eliminating one of the four amino acids Phe.sup.7 -(D)Trp.sup.8 -Lys.sup.9
-Thr.sup.10 with the assumption that such analogs would be more selective.
The somatostatin analog, MK-678:
cyclo(N-Me-Ala.sup.7 -Tyr.sup.7 -(D)Trp.sup.8 -Lys.sup.9 -Val.sup.10 -Phe)
is an example of a highly potent somatostatin analog designed using the
first three approaches above (Lymangrover, et al., Life Science, 34:371,
1984). In this hexapeptide analog, a cis-amide bond is located between
N-Me-Ala and Phe.sup.11, Tyr.sup.7 and Val.sup.10 replace Phe.sup.7 and
Thr.sup.10 respectively, and Phe.sup.11 is incorporated from natural
somatostatin.
Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518 and
4,235,886) includes octreotide:
##STR2##
the only somatostatin analog currently available. It was developed using
the third approach described above. Here, (D)Phe.sup.5 and the reduced
C-terminal Thr.sup.12 -CH.sub.2 OH are assumed to occupy some of the
conformational space available to the natural Phe.sup.6 and Thr.sup.12,
respectively.
The compound TT2-32:
##STR3##
is closely related to octreotide and is an example of implementing the
fourth approach described above. The Lack of Thr.sup.10 is probably
responsible for its high selectivity in terms of antitumor activity.
These examples of highly potent somatostatin analogs indicate that the
phenylalanines in positions 6 and 11 not only play an important role in
stabilizing the pharmacophore conformation but also have a functional role
in the interaction with the receptor. It is still an open question whether
one phenylalanine (either Phe.sup.6 or Phe.sup.11) is sufficient for the
interaction with the receptor or whether both are needed.
It is now known that the somatostatin receptors constitute a family of five
different receptor subtypes (Bell and Reisine, Trends Neurosci., 16,
34-38, 1993), which may be distinguished on the basis of their tissue
specificity and/or biological activity. Somatostatin analogs known in the
art may not provide sufficient selectivity or receptor subtype
selectivity, particularly as anti-neoplastic agents (Reubi and Laissue,
TIPS, 16, 110-115, 1995).
Symptoms associated with metastatic carcinoid tumors (flushing and
diarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas
(watery diarrhea) are treated with somatostatin analogs. Somatostatin has
been also approved for the treatment of severe gastrointestinal
hemorrhages. Somatostatin may also be useful in the palliative treatment
of other hormone-secreting tumors (e.g., pancreatic islet-cell tumors and
acromegaly) and hormone dependent tumors (e.g., chondrosarcoma and
osteosarcoma) due to its anti-secretory activity.
Another important peptide, Bradykinin, is a naturally occurring
nonapeptide, Arg.sup.1 -Pro.sup.2 -Pro.sup.3 -Gly.sup.4 -Phe.sup.5
-Ser.sup.6 -Pro.sup.7 -Phe.sup.8 -Arg.sup.9, formed and released from
precursors in the blood in response to inflammatory stimuli. Elevated
levels of bradykinin also appear in other body fluids and tissues in
pathological states such as asthma, septic shock and common cold. No
clinical abnormalities have been associated so far with bradykinin
deficiency which indicates that bradykinin may not play a critical role in
normal physiology.
However, bradykinin mediates its physiological activities by binding to a
specific receptive molecule called the bradykinin receptor. Two such
bradykinin receptors have been identified so far (these are called B1 and
B2 receptors). Subsequent to binding, the bradykinin signal transduction
pathway includes production of prostaglandins and leukotrienes as well as
calcium activation. Through these mediators, bradykinin is involved in
pain, inflammation, allergic reactions and hypotension. Therefore, a
substance that can block the ability of bradykinin to bind to its
receptor, namely a bradykinin antagonist, should have a significant
therapeutic value for one of the following indications: asthma,
inflammation, septic shock, pain, hypotension and allergy.
The analog used herein to exemplify backbone cyclization is:
D-Arg.sup.0 -Arg-R.sup.1 -Hyp.sup.3 -Gly-Phe-R.sup.2 -D-Phe-Phe.sup.7 -Arg
(wherein, R.sup.1 is Pro, R.sup.2 is Ser in native bradykinin). The change
of proline at position 7 of native bradykinin to D-Phe confers antagonist
activity. This compound was described in Steranka, et al., P.N.A.S. U.S.,
85:3245-3249, 1988 and is one of a plethora of candidate sequences for
modification by the current technology, i.e. backbone cyclization. In this
regard, it is worth noting the applications: WO 89/01781, EP-A-0370453 and
EP-A-0334244 which disclose a wide range of candidate structures.
Antagonist peptides on which stability and/or tissue selectivity can be
conferred by appropriate cyclization will be selected from the many such
known sequences.
According to the present invention a novel synthetic approach is disclosed
providing N.sup..alpha. (.omega.(functionalized)alkylene) amino acid
building units that can be used to synthesize novel N.sup..alpha.
-backbone cyclized peptide analogs such as, but not limited to, novel
somatostatin and bradykinin analogs. None of the above-mentioned
references teaches or suggests N.sup..alpha.
-(.omega.(functionalized)alkylene) amino acids or the novel N.sup..alpha.
-backbone cyclized peptide analogs of the present invention.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide backbone cyclized
peptide analogs that comprise peptide sequences which incorporate at least
two building units, each of which contains one nitrogen atom of the
peptide backbone connected to a bridging group as described below. In the
present invention, one or more pairs of the building units is joined
together to form a cyclic structure. Thus, according to one aspect of the
present invention, backbone cyclized peptide analogs are provided that
have the general Formula (I):
##STR4##
wherein: a and b each independently designates an integer from 1 to 8 or
zero; d, e, and f each independently designates an integer from 1 to 10;
(AA) designates an amino acid residue wherein the amino acid residues in
each chain may be the same or different; E represents a hydroxyl group, a
carboxyl protecting group or an amino group, or CO--E can be reduced to
CH.sub.2 --OH; R, R', R", and R'" each designates an amino acid side-chain
such as H, CH.sub.3, etc., optionally bound with a specific protecting
group; and the lines independently designate a bridging group of the
Formula: (i) --X--M--Y--W--Z--; or (ii) --X--M--Z-- wherein: one line may
be absent; M and W are independently selected from the group consisting of
disulfide, amide, thioether, thioesters, imines, ethers and alkenes; and
X, Y and Z are each independently selected from the group consisting of
alkylene, substituted alkylene, arylene, homo- or hetero-cycloalkylene and
substituted cycloalkylene.
In certain preferred embodiments, the CO--E group of Formula (I) is reduced
to a CH.sub.2 OH group.
Another embodiment of the present invention involves N-backbone to side
chain cyclized peptides of the general formula (II):
##STR5##
wherein the substituents are as defined above.
A preferred embodiment of the present invention involves the backbone
cyclized peptide analog of Formulae I or II wherein the line designates a
bridging group of the Formula: --(CH.sub.2).sub.x --M--(CH.sub.2).sub.y
--W--(CH.sub.2).sub.z -- wherein M and W are independently selected from
the group consisting of disulfide, amide, thioether, thioesters, imines,
ethers and alkenes; x and z each independently designates an integer from
1 to 10, and y is zero or an integer of from 1 to 8, with the proviso that
if y is zero, W is absent.
Further preferred are backbone cyclized peptide analogs of the Formula I or
II wherein R and R' are other than H, such as CH.sub.3, (CH.sub.3).sub.2
CH--, (CH.sub.3).sub.2 CHCH.sub.2 --, CH.sub.3 CH.sub.2 CH(CH.sub.3)--,
CH.sub.3 S(CH.sub.2).sub.2 --, HOCH.sub.2 --, CH.sub.3 CH(OH)--,
HSCH.sub.2 --, NH.sub.2 C(.dbd.O)CH.sub.2 --, NH.sub.2
C(.dbd.O)(CH.sub.2).sub.2 --, NH.sub.2 (CH.sub.2).sub.3 --,
HOC(.dbd.O)CH.sub.2 --, HOC(.dbd.O)(CH.sub.2).sub.2 --, NH.sub.2
(CH.sub.2).sub.4 --, C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --,
HO-phenyl-CH.sub.2 --, benzyl, methylindole, and methylimidazole.
A more preferred embodiment of the present invention is directed to
backbone cyclization to stabilize the .beta.-turn conformation of
bradykinin analogs of the general Formula (III):
##STR6##
wherein M is an amide bond, x and z are each independently an integer of 1
to 10, and K is H or an acyl group.
Also more preferred are backbone cyclized peptide analogs of the present
invention comprising bradykinin analogs of the general Formula (IVa):
##STR7##
wherein M is an amide bond, x and z are each independently an integer of 1
to 10, K is H or an acyl group, and R.sup.6 is Gly or Ser; or the general
Formula (IVb):
##STR8##
wherein x is an integer of 1 to 10; K is H or an acyl group; (R.sup.6) is
selected from the group of D-Asp, L-Asp, D-Glu and L-Glu; and z is
according to the amino acid specified: 1 in case of D and L-Asp, and 2 in
the case of D and L Glu.
Further more preferred backbone cyclized peptide analogs according to the
present invention having bradykinin antagonist activity have the Formula
(V):
##STR9##
wherein M is an amide bond, x and z are each independently an integer of 1
to 10, and K is H or an acyl group.
Specifically preferred backbone cyclized peptide analogs of the present
invention are:
1) Ada-(D)Arg-Arg-cyclo(N.sup..alpha.
(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH;
2) H-D-Arg-Arg-cyclo(N.sup..alpha.
(1-(4-propanoyl))Gly-Hyp-Phe-N.sup..alpha.
(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH; and
3) H-D-Arg-Arg-cyclo(N.sup..alpha. (4-propanoyl)Gly-Hyp-Phe-N.sup..alpha.
(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH.
Another preferred aspect of the present invention is directed to backbone
cyclization to generate novel somatostatin analogs linked between
positions 6 and 11, leaving the phenylalanine side chains untouched. This
conformational stabilization is much more rigid than the Phe.sup.6,
Phe.sup.11 hydrophobic interaction in natural somatostatin and is more
stable to reduction/oxidation reactions than the Cys-Cys disulfide bridge.
In other words, for the first time a stable covalent bridge can be
achieved while either one or both of the original Phe.sup.6 and Phe.sup.11
are retained.
Moreover, backbone cyclizations can also be used to anchor the .beta.-turn,
not only in positions 6 and 11 but also inside the active reaction of
Phe.sup.7 -(D)Trp.sup.8 -Lys.sup.9 -Thr.sup.10, yielding either a
monocyclic analog with a preferable conformation or a very rigid bicyclic
analog. Here again, the side chains of the pharmacologically active amino
acids remain untouched and the only change is in limiting the
conformational space.
As used herein and in the claims in the following more preferred backbone
cyclized peptide analogs, the superscript numbers following the amino
acids refer to their position numbers in the native Somatostatin.
A more preferred backbone cyclized peptide novel analog is the Formula
(XIVa):
##STR10##
with a most preferred analog being the Formula (XIVb):
##STR11##
wherein m and n are 1, 2 or 3; X is CH.sub.2 OH or CONH.sub.2 ; R.sup.5 is
absent or is Gly, (D)- or (L)-Ala, Phe, Nal and .beta.-Asp(Ind); R.sup.6
and R.sup.11 are independently Gly or (D)- or (L)-Phe; R.sup.7 is Phe or
Tyr; R.sup.10 is absent or is Gly, Abu, Thr or Val; R.sup.12 is absent or
is Thr or Nal, and Y.sup.2 is selected from the group consisting of amide,
disulfide, thioether, imines, ethers and alkenes. In these monocyclic
somatostatin analogs, a backbone cyclization replaces the Cys.sup.6
-Cys.sup.11 disulfide bridge, leaving the phenylalanine side chains as in
the natural somatostatin. Still more preferred is the analog wherein
Phe.sup.7 is replaced with Tyr.sup.7 and Thr.sup.10 is replaced with
Val.sup.10.
Other more preferred monocyclic analogs that anchor the molecule in
positions inside the active region rather than in positions 6 and 11 are
formulae XV (a and b) and XVI (a-c):
##STR12##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or CONH.sub.2
; R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind);
R.sup.6 is (D) or (L)-Phe; R.sup.10 is absent or is Gly, Abu or Thr; and
R.sup.11 is (D)- or (L)-Phe; R.sup.12 is absent or is Thr or Nal, and
Y.sup.1 is selected from the group consisting of amide, disulfide,
thioether, imines, ethers and alkenes.
Still other more preferred analogs incorporate backbone cyclization in
positions 6 and 11 as in Formula XIV, together with the backbone
cyclizations as in Formula XV and XVI, yielding rigid bicyclic analogs of
the Formulae XVII (a and b) and XVIII (a and b):
##STR13##
wherein i, j, m and n are independently 1, 2 or 3; X is CH.sub.2 OH or
CONH.sub.2 ; R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or
.beta.-Asp(Ind); R.sup.6 and R.sup.11 are independently Gly or (D)- or
(L)-Phe; R.sup.10 is absent or is Gly, Abu, Val or Thr; R.sup.12 is absent
or is Thr or Nal; and Y.sup.1 and Y.sup.2 are independently selected from
the group consisting of amide, disulfide, thioether, imines, ethers and
alkenes.
Other more preferred bicyclic analogs differ from Formulae XVII and XVIII
by the replacement of the amino acids at positions 6 and 11 by cysteines
which form a disulfide bond, leaving only one backbone cyclization in the
Formulae XIX (a and b) and XX (a and b):
##STR14##
wherein i and j are independently 1, 2 or 3; X is CH.sub.2 OH or NH.sub.2 ;
R.sup.5 is absent or is (D)- or (L)-Phe, Nal, or .beta.-Asp(Ind); R.sup.6
and R.sup.11 are independently Gly or Phe; R.sup.10 is absent or is Gly,
Abu or Thr; R.sup.12 is absent or is Thr or Nal; and Y.sup.1 is selected
from the group consisting of amide, disulfide, thioether, imines, ethers
and alkenes.
Another aspect of the present invention is a method for the preparation of
cyclic peptides of the general Formula (I):
##STR15##
wherein: a and b each independently designates an integer from 1 to 8 or
zero; d, e, and f each independently designates an integer from 1 to 10;
(AA) designates an amino acid residue wherein the amino acid residues in
each chain may be the same or different; E represents a hydroxyl group, a
carboxyl protecting group or an amino group, or CO--E can be reduced to
CH.sub.2 --OH; R, R', R", and R'" each designates an amino acid side-chain
optionally bound with a specific protecting group; and the lines designate
a bridging group of the Formula:
(i) --X--M--Y--W--Z--; or (ii) --X--M--Z--
wherein: one line may be absent; M and W are independently selected from
the group consisting of disulfide, amide, thioether, thioesters, imines,
ethers and alkenes; and X, Y and Z are each independently selected from
the group consisting of alkylene, substituted alkylene, arylene, homo- or
hetero-cycloalkylene and substituted cycloalkylene. This method comprises
the steps of incorporating at least one N.sup..alpha.
-.omega.-functionalized derivative of amino acids of Formula (VI):
##STR16##
wherein X is a spacer group selected from the group consisting of alkylene,
substituted alkylene, arylene, cycloalkylene and substituted
cycloalkylene; R' is an amino acid side chain, optionally bound with a
specific protecting group; B is a protecting group selected from the group
consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls; and G is
a functional group selected from the group consisting of amines, thiols,
alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl
halides; and A is a specific protecting group of G; into a peptide
sequence and subsequently selectively cyclizing the functional group with
one of the side chains of the amino acids in said peptide sequence or with
another .omega.-functionalized amino acid derivative.
A further object of the present invention is directed to building units
known as a N.sup..alpha. -.omega.-functionalized derivatives of the
general Formula (VI) of amino acids which are prerequisites for the
cyclization process:
##STR17##
wherein X is a spacer group selected from the group consisting of alkylene,
substituted alkylene, arylene, cycloalkylene and substituted
cycloalkylene; R is the side chain of an amino acid, optionally bound with
a specific protecting group; B is a protecting group selected from the
group consisting of alkyloxy, substituted alkyloxy, or aryloxy carbonyls;
and G is a functional group selected from the group consisting of amines,
thiols, alcohols, carboxylic acids and esters, aldehydes and alkyl
halides; and A is a protecting group thereof.
Preferred building units are the .omega.-functionalized amino acid
derivatives wherein X is alkylene; G is a thiol group, an amine group or a
carboxyl group; R is phenyl, methyl or isobutyl; with the proviso that
when G is an amine group, R is other than H.
Further preferred are .omega.-functionalized amino acid derivatives wherein
R is protected with a specific protecting group.
More preferred are .omega.-functionalized amino acid derivatives of the
Formulae:
##STR18##
wherein X, R, A and B are as defined above.
Specifically preferred .omega.-functionalized amino acid derivatives
include the following:
1) N.sup..alpha. -(Fmoc)(3-Boc-amino propylene)-(S)Phenylalanine;
2) N.sup..alpha. -(Fmoc)(3-Boc-amino propylene)-(R)Phenylalanine;
3) N.sup..alpha. -(Fmoc)(4-Boc-amino butylene)-(S)Phenylalanine;
4) N.sup..alpha. -(Fmoc)(3-Boc-amino propylene)-(S)Alanine;
5) N.sup..alpha. -(Fmoc)(6-Boc-amino hexylene)-(S)Alanine;
6) N.sup..alpha. -(Fmoc)(3-Boc-amino propylene)-(R)Alanine;
7) N.sup..alpha. -(2-(benzylthio)ethylene)glycine ethyl ester;
8) N.sup..alpha. -(2-(benzylthio)ethylene)(S)leucine methyl ester;
9) N.sup..alpha. -(3-(benzylthio)propylene)(S)leucine methyl ester:
10) Boc-N.sup..alpha. -(2-(benzylthio)ethylene)glycine;
11) Boc-N.sup..alpha. -(2-(benzylthio)ethylene)(S)phenylalanine;
12) Boc-N.sup..alpha. -(3-(benzylthio)propylene)(S)phenylalanine;
13) Boc-L-phenylalanyl-N.sup..alpha. -(2-(benzylthio)ethylene)glycine-ethyl
ester;
14) Boc-L-phenylalanyl-N.sup..alpha.
-(2-(benzylthio)ethylene)-(S)phenylalanine methyl ester;
15) N.sup..alpha. (Fmoc)-(2-t-butyl carboxy ethylene)glycine;
16) N.sup..alpha. (Fmoc)-(3-t-butyl carboxy propylene)glycine;
17) N.sup..alpha. (Fmoc)(2-t-butyl carboxy ethylene)(S)phenylalanine;
18) N.sup..alpha. (Fmoc)(2-Boc amino ethylene)glycine;
19) N.sup..alpha. (Fmoc)(3-Boc amino propylene)glycine;
20) N.sup..alpha. (Fmoc)(4-Boc amino butylene)glycine; and
21) N.sup..alpha. (Fmoc)(6-Boc amino hexylene)glycine.
Novel, practical, generally applicable processes for the preparation of
these N.sup..alpha. -.omega.-functionalized derivatives of amino acids are
a further aspect of this invention.
As such, an object of this invention is a method of making an
.omega.-functionalized amino acid derivative of the general Formula:
##STR19##
wherein X is a spacer group selected from the group consisting of alkylene,
substituted alkylene, arylene, cycloalkylene and substituted
cycloalkylene; R is the side chain of an amino acid, such as H, CH.sub.3,
etc.; A and B are protecting groups selected from the group consisting of
alkyloxy, substituted alkyloxy, or aryloxy carbonyls;
comprising the steps of:
i) reacting a diamine compound of the general Formula:
##STR20##
wherein A, B and X are as defined above,
with a triflate of Formula CF.sub.3 SO.sub.2 --O--CH(R)--CO--E wherein E is
a carboxyl protecting group and R is as defined above; to yield a compound
of Formula:
##STR21##
wherein A, B, E, R and X are as defined above
ii) and deprotecting the carboxyl to yield an
N.sup..alpha..omega.-functionalized amino acid derivative, wherein the
.omega.-functional group is an amine.
A further object of this invention is a method of making an
.omega.-functionalized amino acid derivative of the general Formula:
##STR22##
where B is a protecting group selected from the group of substituted
alkyloxy, substituted alkyloxy, or aryloxy carbonyls; R is the side chain
of an amino acid, such as H, CH.sub.3, etc.; X is a spacer group selected
from the group of alkylene, substituted alkylene, arylene, cycloalkylene
or substituted cycloalkylene; and A is a protecting group selected from
the group of alkyl or substituted alkyl, thio ethers or aryl or
substituted aryl thio ethers;
comprising the steps of:
i) reacting a compound of the general Formula B--NH--X--S--A with a
triflate of the general Formula CF.sub.3 SO.sub.2 --O--CH(R)--CO--E
wherein E is a carboxyl protecting group and A, X and R are as defined
above, to give a compound of the Formula:
##STR23##
ii) selectively removing the protecting group E, and
iii) protecting the free amino group to yield an N.sup..alpha.
(.omega.-functionalized) amino acid derivative, wherein the
.omega.-functional group is a thiol.
A further object of this invention is a method of making an
.omega.-functionalized amino acid derivative of the general Formula:
##STR24##
where B is a protecting group selected from the group of alkyloxy,
substituted alkyloxy, or aryloxy carbonyls; R is the side chain of an
amino acid, such as H, CH.sub.3, etc.; X is a spacer group selected from
the group of alkylene, substituted alkylene, arylene, cycloalkylene or
substituted cycloalkylene; and A is a protecting group selected from the
group of alkyl or substituted alkyl, esters, or thio esters or substituted
aryl esters or thio esters;
comprising the steps of:
i) reacting a compound of the general Formula B--NH--X--CO--A with a
triflate of the general Formula CF.sub.3 SO.sub.2 --O--CH(R)--CO--E
wherein E is a carboxyl protecting group and A, B, X and R are as defined
above, to give a compound of Formula:
##STR25##
ii) and selectively removing protecting group E, to yield an N.sup..alpha.
(.omega.-functionalized) amino acid derivative, wherein the
.omega.-functional group is a carboxyl.
A further aspect of this invention is to provide methods for the
preparation of novel backbone cyclic peptides, comprising the steps of
incorporating at least one N.sup..alpha. -.omega.-functionalized
derivatives of amino acids into a peptide sequence and subsequently
selectively cyclizing the functional group with one of the side chains of
the amino acids in said peptide sequence, or with another
.omega.-functionalized amino acid derivative.
Backbone cyclized analogs of the present invention may be used as
pharmaceutical compositions and for methods for the treatment of disorders
including: acute asthma, septic shock, brain trauma and other traumatic
injury, post-surgical pain, all types of inflammation, cancers, endocrine
disorders and gastrointestinal disorders.
Therefore, further objects of the present invention are directed to
pharmaceutical compositions comprising pharmacologically active backbone
cyclized peptide agonists and antagonists prepared according to the
methods disclosed herein and a pharmaceutically acceptable carrier or
diluent; and methods for the treatment of inflammation, septic shock,
cancer or endocrine disorders and gastrointestinal disorders therewith.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing in vitro biostability of somatostatin and three
analogs thereof in human serum. The graph depicts the percentage of
undegraded molecules for each of the compounds initially and after various
periods of time.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
All abbreviations used are in accordance with the IUPAC-IUB recommendations
on Biochemical Nomenclature (J. Biol. Chem., 247:977-983, 1972) and later
supplements.
As used herein and in the claims, the phrase "an amino acid side chain"
refers to the distinguishing substituent attached to the .alpha.-carbon of
an amino acid; such distinguishing groups are well known to those skilled
in the art. For instance, for the amino acid glycine, the R group is H;
for the amino acid alanine, R is CH.sub.3, and so on. Other typical side
chains of amino acids include the groups: (CH.sub.3).sub.2 CH--,
(CH.sub.3).sub.2 CHCH.sub.2 --, CH.sub.3 CH.sub.2 CH(CH.sub.3)--, CH.sub.3
S(CH.sub.2).sub.2 --, HOCH.sub.2 --, CH.sub.3 CH(OH)--, HSCH.sub.2 --,
NH.sub.2 C(.dbd.O)CH.sub.2 --, NH.sub.2 C(.dbd.O)(CH.sub.2).sub.2 --,
NH.sub.2 (CH.sub.2).sub.3 --, HOC(.dbd.O)CH.sub.2 --,
HOC(.dbd.O)(CH.sub.2).sub.2 --, NH.sub.2 (CH.sub.2).sub.4 --,
C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --, HO-phenyl-CH.sub.2 --, benzyl,
methylindole, and methylimidazole.
As used herein and in the claims, the letters "(AA)" and the term "amino
acid" are intended to include common natural or synthetic amino acids, and
common derivatives thereof, known to those skilled in the art, including
but not limited to the following. Typical amino-acid symbols denote the L
configuration unless otherwise indicated by D appearing before the symbol.
Abbreviated Designation Amino Acids
Abu .alpha.-Amino butyric acid
Ala L-Alanine
Arg L-Arginine
Asn L-Asparagine
Asp L-Aspartic acid
.beta.Asp(Ind) .beta.-Indolinyl aspartic acid
Cys L-Cysteine
Glu L-Glutamic acid
Gln L-Glutamine
Gly Glycine
His L-Histidine
Hyp trans-4-L-Hydroxy Proline
Ile L-Isoleucine
Leu L-Leucine
Lys L-Lysine
Met L-Methionine
Nal .beta.-Naphthyl alanine
Orn Ornithine
Phe L-Phenylalanine
Pro L-Proline
Ser L-Serine
Thr L-Threonine
Trp L-Tryptophane
Tyr L-Tyrosine
Val L-Valine
Typical protecting groups, coupling agents, reagents and solvents such as
but not limited to those listed below have the following abbreviations as
used herein and in the claims. One skill in the art would understand that
the compounds listed within each group may be used interchangeably; for
instance, a compound listed under "reagents and solvents" may be used as a
protecting group, and so on. Further, one skill in the art would know
other possible protecting groups, coupling agents and reagents/solvents;
these are intended to be within the scope of this invention.
Abbreviated Designation Protecting Groups
Ada Adamantane acetyl
Alloc Allyloxycarbonyl
Allyl Allyl ester
Boc tert-butyloxycarbonyl
Bzl Benzyl
Fmoc Fluorenylmethyloxycarbonyl
OBzl Benzyl ester
OEt Ethyl ester
OMe Methyl ester
Tos (Tosyl) p-Toluenesulfonyl
Trt Triphenylmethyl
Z Benzyloxycarbonyl
Abbreviated Designation Coupling Agents
BOP Benzotriazol-1-yloxytris-
(dimethyl-amino) phosphonium
hexafluorophosphate
DIC Diisopropylcarbodiimide
HBTU 2-(1H-Benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium
hexafluorophosphate
PyBrOP Bromotripyrrolidinophosphonium
hexafluorophosphate
PyBOP Benzotriazol-1-yl-oxy-tris-
pyrrolidino-phosphonium
hexafluorophosphate
TBTU O-(1,2-dihydro-2-oxo-1-pyridyl)-
N,N,N',N'-tetramethyluronium
tetrafluoroborate
Reagents
Abbreviated Designation and Solvents
ACN Acetonitrile
AcOH Acetic acid
Ac.sub.2 O Acetic acid anhydride
AdacOH Adamantane acetic acid
Alloc-Cl Allyloxycarbonyl chloride
Boc.sub.2 O Di-tert butyl dicarbonate
DMA Dimethylacetamide
DMF N,N-dimethylformamide
DIEA Diisopropylethylamine
Et.sub.3 N Triethylamine
EtOAc Ethyl acetate
FmocOSu 9-fluorenylmethyloxy carbonyl
N-hydroxysuccinimide ester
HOBT 1-Hydroxybenzotriazole
HF Hydrofluoric acid
MeOH Methanol
Mes (Mesyl) Methanesulfonyl
NMP 1-methyl-2-pyrrolidinone
nin. Ninhydrin
i-PrOH Iso-propanol
Pip Piperidine
PP 4-pyrrolidinopyridine
Pyr Pyridine
SRIF Somatotropin release inhibiting
factor
SST Somatostatin
SSTR Somatostatin receptor
TEA Triethylamine
TFA Trifluoroacetic acid
THF Tetrahydrofuran
Triflate (Trf) Trifluoromethanesulfonyl
Trf.sub.2 O Trifluoromethanesulfonic acid
anhydride
The compounds herein described may have asymmetric centers. All chiral,
diastereomeric, and racemic forms are included in the present invention.
Many geometric isomers of olefins and the like can also be present in the
compounds described herein, and all such stable isomers are contemplated
in the present invention.
By "stable compound" or "stable structure" is meant herein a compound that
is sufficiently robust to survive isolation to a useful degree of purity
from a reaction mixture, and Formulation into an efficacious therapeutic
agent.
As used herein and in the claims, "alkyl" or "alkylenyl" is intended to
include both branched and straight-chain saturated aliphatic hydrocarbon
groups having one to ten carbon atoms; "alkenyl" is intended to include
hydrocarbon chains of either a straight or branched configuration and one
or more unsaturated carbon-carbon bonds which may occur in any stable
point along the chain, such as ethenyl, propenyl, and the like; and
"alkynyl" is intended to include hydrocarbon chains of either a straight
or branched configuration and one or more triple carbon-carbon bonds which
may occur in any stable point along the chain, such as ethynyl, propynyl,
and the like.
As used herein and in the claims, "aryl" is intended to mean any stable 5-
to 7-membered monocyclic or bicyclic or 7- to 14-membered bicyclic or
tricyclic carbon ring, any of which may be saturated, partially
unsaturated or aromatic, for example, phenyl, naphthyl, indanyl, or
tetrahydronaphthyl tetralin, etc.
As used herein and in the claims, "alkyl halide" is intended to include
both branched and straight-chain saturated aliphatic hydrocarbon groups
having one to ten carbon atoms, wherein 1 to 3 hydrogen atoms have been
replaced by a halogen atom such as Cl, F, Br, and I.
As used herein and in the claims, the term "heterocyclic" is intended to
mean any stable 5- to 7-membered monocyclic or bicyclic or 7- to
10-membered bicyclic heterocyclic ring, which is either saturated or
unsaturated, and which consists of carbon atoms and from 1 to 3
heteroatoms selected from the group consisting of N, O and S and wherein
the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen
atom optionally be quaternized, and including any bicyclic group in which
any of the above-defined heterocyclic rings is fused to a benzene ring.
The heterocyclic ring may be attached to its pendant group at any
heteroatom or carbon atom which results in a stable structure. The
heterocyclic rings described herein may be substituted on carbon or on a
nitrogen atom if the resulting compound is stable. Examples of such
heterocycles include, but are not limited to pyridyl, pyrimidinyl,
furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,
benzofuranyl, benzothiophenyl, indolyl, indolenyl, quinolinyl,
piperidonyl, pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, or
octahydroisoquinolinyl and the like.
As used herein and in the claims, the phrase "therapeutically effective
amount" means that amount of novel backbone cyclized peptide analog or
composition comprising same to administer to a host to achieve the desired
results for the indications described herein, such as but not limited of
inflammation, septic shock, cancer, endocrine disorders and
gastrointestinal disorders.
The term, "substituted" as used herein and in the claims, means that any
one or more hydrogen atoms on the designated atom is replaced with a
selection from the indicated group, provided that the designated atom's
normal valency is not exceeded, and that the substitution results in a
stable compound.
When any variable (for example R, x, z, etc.) occurs more than one time in
any constituent or in Formulae (I to XX) or any other Formula herein, its
definition on each occurrence is independent of its definition at every
other occurrence. Also, combinations of substituents and/or variables are
permissible only if such combinations result in stable compounds.
Synthetic Approach
According to the present invention peptide analogs are cyclized via
bridging groups attached to the alpha nitrogens of amino acids that permit
novel non-peptidic linkages. In general, the procedures utilized to
construct such peptide analogs from their building units rely on the known
principles of peptide synthesis; most conveniently, the procedures can be
performed according to the known principles of solid phase peptide
synthesis. The innovation requires replacement of one or more of the amino
acids in a peptide sequence by novel building units of the general
Formula:
##STR26##
wherein R is the side chain of an amino acid, X is a spacer group and G is
the functional end group by means of which cyclization will be effected.
The side chain R is the side chain of any natural or synthetic amino acid
that is selected to be incorporated into the peptide sequence of choice. X
is a spacer group that is selected to provide a greater or lesser degree
of flexibility in order to achieve the appropriate conformational
constraints of the peptide analog. Such spacer groups include alkylene
chains, substituted, branched and unsaturated alkylenes, arylenes,
cycloalkylenes, unsaturated and substituted cycloakylenes. Furthermore, X
and R can be combined to form a heterocyclic structure.
A preferred embodiment of the present invention utilizes alkylene chains
containing from two to ten carbon atoms.
The terminal (.omega.) functional groups to be used for cyclization of the
peptide analog include but are not limited to:
a. Amines, for reaction with electrophiles such as activated carboxyl
groups, aldehydes and ketones (with or without subsequent reduction), and
alkyl or substituted alkyl halides.
b. Alcohols, for reaction with electrophiles such as activated carboxyl
groups.
c. Thiols, for the formation of disulfide bonds and reaction with
electrophiles such as activated carboxyl groups, and alkyl or substituted
alkyl halides.
d. 1,2 and 1,3 Diols, for the formation of acetals and ketals.
e. Alkynes or Substituted Alkynes, for reaction with nucleophiles such as
amines, thiols or carbanions; free radicals; electrophiles such as
aldehydes and ketones, and alkyl or substituted alkyl halides; or
organometallic complexes.
f. Carboxylic Acids and Esters, for reaction with nucleophiles (with or
without prior activation), such as amines, alcohols, and thiols.
g. Alkyl or Substituted Alkyl Halides or Esters, for reaction with
nucleophiles such as amines, alcohols, thiols, and carbanions (from active
methylene groups such as acetoacetates or malonates); and formation of
free radicals for subsequent reaction with alkenes or substituted alkenes,
and alkynes or substituted alkynes.
h. Alkyl or Aryl Aldehydes and Ketones for reaction with nucleophiles such
as amines (with or without subsequent reduction), carbanions (from active
methylene groups such as acetoacetates or malonates), diols (for the
formation of acetals and ketals).
i. Alkenes or Substituted Alkenes, for reaction with nucleophiles such as
amines, thiols, carbanions, free radicals, or organometallic complexes.
j. Active Methylene Groups, such as malonate esters, acetoacetate esters,
and others for reaction with electrophiles such as aldehydes and ketones,
alkyl or substituted alkyl halides.
It will be appreciated that during synthesis of the peptide these reactive
end groups, as well as any reactive side chains, must be protected by
suitable protecting groups. Suitable protecting groups for amines are
alkyloxy, substituted alkyloxy, and aryloxy carbonyls including, but not
limited to, tert butyloxycarbonyl (Boc), Fluorenylmethyloxycarbonyl
(Fmoc), Allyloxycarbonyl (Alloc) and Benzyloxycarbonyl (Z).
Carboxylic end groups for cyclizations may be protected as their alkyl or
substituted alkyl esters or thio esters or aryl or substituted aryl esters
or thio esters. Examples include but are not limited to tertiary butyl
ester, allyl ester, benzyl ester, 2-(trimethylsilyl)ethyl ester and
9-methyl fluorenyl.
Thiol groups for cyclizations may be protected as their alkyl or
substituted alkyl thio ethers or disulfides or aryl or substituted aryl
thio ethers or disulfides. Examples of such groups include but are not
limited to tertiary butyl, trityl(triphenylmethyl), benzyl,
2-(trimethylsilyl)ethyl, pixyl(9-phenylxanthen-9-yl), acetamidomethyl,
carboxy-methyl, 2-thio-4-nitropyridyl.
It will further be appreciated by the artisan that the various reactive
moieties will be protected by different protecting groups to allow their
selective removal. Thus, a particular amino acid will be coupled to its
neighbor in the peptide sequence when the N.sup..alpha. is protected by,
for instance, protecting group A. If an amine is to be used as an end
group for cyclization in the reaction scheme the N.sup..omega. will be
protected by protecting group B, or an .epsilon. amino group of any lysine
in the sequence will be protected by protecting group C, and so on.
The coupling of the amino acids to one another is performed as a series of
reactions as is known in the art of peptide synthesis. Novel building
units of the invention, namely the N.sup..alpha. -.omega. functionalized
amino acid derivatives are incorporated into the peptide sequence to
replace one or more of the amino acids. If only one such N.sup..alpha.
-.omega. functionalized amino acid derivative is selected, it will be
cyclized to a side chain of another amino acid in the sequence. For
instance: (a) an N.sup..alpha. -(.omega.-amino alkylene) amino acid can be
linked to the carboxyl group of an aspartic or glutamic acid residue; (b)
an N.sup..alpha. -(.omega.-carboxylic alkylene) amino acid can be linked
to the .epsilon.-amino group of a lysine residue; (c) an N.sup..alpha.
-(.omega.-thio alkylene) amino acid can be linked to the thiol group of a
cysteine residue; and so on. A more preferred embodiment of the invention
incorporates two such N.sup..alpha. -.omega.-functionalized amino acid
derivatives which may be linked to one another to form N-backbone to
N-backbone cyclic peptide analogs. Three or more such building units can
be incorporated into a peptide sequence to create bi-cyclic peptide
analogs as will be elaborated below. Thus, peptide analogs can be
constructed with two or more cyclizations, including N-backbone to
N-backbone, as well as backbone to side-chain or any other peptide
cyclization.
As stated above, the procedures utilized to construct peptide analogs of
the present invention from novel building units generally rely on the
known principles of peptide synthesis. However, it will be appreciated
that accommodation of the procedures to the bulkier building units of the
present invention may be required. Coupling of the amino acids in solid
phase peptide chemistry can be achieved by means of a coupling agent such
as but not limited to dicyclohexycarbodiimide (DCC),
bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP--Cl),
benzotriazolyl-N-oxytrisdimethyl-aminophosphonium hexafluoro phosphate
(BOP), 1-oxo-1-chlorophospholane (Cpt--Cl), hydroxybenzotriazole (HOBT),
or mixtures thereof.
It has now been found that coupling of the bulky building units of the
present invention may require the use of additional coupling reagents
including, but not limited to: coupling reagents such as PyBOP.RTM.
(Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate),
PyBrOP.RTM. (Bromo-tris-pyrrolidino-phosphonium hexafluoro-phosphate),
HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate), TBTU
(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate).
Novel coupling chemistries may be used, such as pre-formed
urethane-protected N-carboxy anhydrides (UNCA's) and pre-formed acyl
fluorides. Said coupling may take place at room temperature and also at
elevated temperatures, in solvents such as toluene, DCM (dichloromethane),
DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl
pyrrolidinone) or mixtures of the above.
One object of the present invention is a method for the preparation of
backbone cyclized peptide analogs of Formula (I):
##STR27##
wherein the substituents are as defined above;
comprising the steps of incorporating at least one N.sup..alpha.
-.omega.-functionalized derivatives of amino acids of Formula (VI):
##STR28##
wherein X is a spacer group selected from the group consisting of alkylene,
substituted alkylene, arylene, cycloalkylene and substituted
cycloalkylene; R' is an amino acid side chain such as H, CH.sub.3, etc.,
optionally bound with a specific protecting group; B is a protecting group
selected from the group consisting of alkyloxy, substituted alkyloxy, or
aryloxy carbonyls; and G is a functional group selected from the group
consisting of amines, thiols, alcohols, carboxylic acids and esters,
aldehydes, alcohols and alkyl halides; and A is a specific protecting
group of G;
with a compound of the Formula (VII):
H.sub.2 N--(AA).sub.f --CO--E Formula (VII)
wherein f is an integer from 1 to 10; (AA) designates an amino acid residue
wherein the amino acid residues may be the same or different, and E is a
hydroxyl, a carboxyl protecting group or an amide to give a compound of
the general Formula:
##STR29##
(ii) selectively removing protecting group B and reacting the unprotected
compound with a compound of Formula:
B--NH--(AA).sub.e --COOH Formula (IX)
wherein B and (AA) are as described above and e is an integer from 1 to 10,
to give a compound of Formula:
##STR30##
wherein B, (AA), e, R.sup.1, and f are as described above;
(iii) removing the protecting group B from the compound of the Formula (X)
and reacting the unprotected compound with a compound of Formula:
##STR31##
wherein X' is a spacer group selected from the group consisting of
alkylenes, substituted alkylenes, arylenes, cycloalkylenes and substituted
alkylenes; G' is a functional group selected from amines, thiols,
carboxyls, aldehydes or alcohols; A' is a specific-protecting group
thereof; R.sup.1 is an amino acid side chain such as H, CH.sub.3, etc.,
optionally bound with a specific protecting group; and B is a protecting
group;
to yield a compound of Formula:
##STR32##
(iv) removing the protecting group B and reacting the unprotected compound
with a compound of Formula:
B--NH--(AA).sub.d --COOH Formula (IXa)
to yield a compound of Formula:
##STR33##
(v) selectively removing protecting groups A and A' and reacting the
terminal groups G and G' to form a compound of the Formula:
##STR34##
wherein d, e and f are independently an integer from 1 to 10; (AA) is an
amino acid residue wherein the amino acid residues in each chain may be
the same or different; E is an hydroxyl group, a carboxyl protecting group
or an amino group; R and R' are independently an amino acid side-chain
such as H, CH.sub.3, etc.; and the line designates a bridging group of the
Formula: --X--M--Y--W--Z--
wherein M and W are independently selected from the group consisting of
disulfide, amide, thioether, imine, ether, and alkene; X, Y and Z are
independently selected from the group consisting of alkylene, substituted
alkylene, arylene, cycloalkylene, and substituted cycloalkylene;
(vi) removing all remaining protecting groups to yield a compound of
Formula (I).
Bicyclic analogs are prepared in the same manner, that is, by repetition of
steps (v) and (vi). The determination of which residues are cyclized with
which other residues is made through the choice of blocking groups. The
various blocking groups may be removed selectively, thereby exposing the
selected reactive groups for cyclization.
Preferred are methods for the preparation of backbone cyclized peptide
analogs of Formula (I) wherein G is an amine, thiol or carboxyl group; R
and R.sup.1 are each other than H, such as CH.sub.3, (CH.sub.3).sub.2
CH--, (CH.sub.3).sub.2 CHCH.sub.2 --, CH.sub.3 CH.sub.2 CH(CH.sub.3)--,
CH.sub.3 S(CH.sub.2).sub.2 --, HOCH.sub.2 --, CH.sub.3 CH(OH)--,
HSCH.sub.2 --, NH.sub.2 C(.dbd.O)CH.sub.2 --, NH.sub.2 C(.dbd.O)
(CH.sub.2).sub.2 --, HOC(.dbd.O)CH.sub.2 --, HOC(.dbd.O) (CH.sub.2).sub.2
--, NH.sub.2 (CH.sub.2).sub.4 --, C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --,
HO-phenyl-CH.sub.2 --, benzyl, methylindole, and methylimidazole, and
wherein E is covalently bound to an insoluble polymeric support.
Another object of the present invention is a method for the preparation of
backbone cyclized peptide analogs of Formula (II):
##STR35##
wherein the substituents are as defined above;
comprising the steps of: incorporating at least one .omega.-functionalized
amino acid derivative of the general Formula (VI):
##STR36##
wherein X is a spacer group selected from the group consisting of alkylene,
substituted alkylene, arylene, cycloalkylene and substituted
cycloalkylene; R is the side chain of an amino acid, such as H, CH.sub.3,
etc.; B is a protecting group selected from the group consisting of
alkyloxy, substituted alkyloxy, or aryloxy carbonyls; and G is a
functional group selected from the group consisting of amines, thiols,
alcohols, carboxylic acids and esters or alkyl halides and A is a
protecting group thereof;
into a peptide sequence and subsequently selectively cyclizing the
functional group with one of the side chains of the amino acids in said
peptide sequence.
Preferred is the method for the preparation of backbone cyclized peptide
analogs of Formula (II) wherein G is a carboxyl group or a thiol group; R
is CH.sub.3, (CH.sub.3).sub.2 CH--, (CH.sub.3).sub.2 CHCH.sub.2 --,
CH.sub.3 CH.sub.2 CH(CH.sub.3)--, CH.sub.3 S(CH.sub.2).sub.2 --,
HOCH.sub.2 --, CH.sub.3 CH(OH)--, HSCH.sub.2 --, NH.sub.2
C(.dbd.O)CH.sub.2 --, NH.sub.2 C(.dbd.O)(CH.sub.2).sub.2 --,
HOC(.dbd.O)CH.sub.2 --, HOC(.dbd.O)(CH.sub.2).sub.2 --, NH.sub.2
(CH.sub.2).sub.4 --, C(NH.sub.2).sub.2 NH(CH.sub.2).sub.3 --,
HO-phenyl-CH.sub.2 --, benzyl, methylindole, and methylimidazole, and
wherein E is covalently bound to an insoluble polymeric support.
Preparation of backbone to side chain cyclized peptide analogs is
exemplified in Scheme I below. In this schematic example, the bridging
group consists of alkylene spacers and an amide bond formed between an
acidic amino acid side chain (e.g. aspartic or glutamic acid) and an
.omega.-functionalized amino acid having a terminal amine.
(Scheme I)
Preparation of Peptides with Backbone to Side Chain Cyclization
One preferred procedure for preparing the desired backbone cyclic peptides
involves the stepwise synthesis of the linear peptides on a solid support
and the backbone cyclization of the peptide either on the solid support or
after removal from the support. The C-terminal amino acid is bound
covalently to an insoluble polymeric support by a carboxylic acid ester or
other linkages such as amides. An example of such support is a
polystyrene-co-divinyl benzene resin. The polymeric supports used are
those compatible with such chemistries as Fmoc and Boc and include for
example PAM resin, HMP resin and chloromethylated resin. The resin bound
amino acid is deprotected for example with TFA to give (1) below and to it
is coupled the second amino acid, protected on the N.sup..alpha. for
example by Fmoc, using a coupling reagent like BOP. The second amino acid
is deprotected to give (3) using for example piperidine 20% in DMF. The
subsequent protected amino acids can then be coupled and deprotected at
ambient temperature. After several cycles of coupling and deprotection
that gives peptide (4), an amino acid having for example carboxy side
chain is coupled to the desired peptide. One such amino acid is
Fmoc-aspartic acid t-butyl ester. After deprotection of the N.sup..alpha.
Fmoc protecting group that gives peptide (5), the peptide is again
elongated by methods well known in the art to give (6). After deprotection
a building unit for backbone cyclization (the preparation of which is
described in Schemes III-VIII) is coupled to the peptide resin using for
example the coupling reagent BOP to give (7). One such building unit is
for example Fmoc-N.sup..alpha. (.omega.-Boc-amino alkylene)amino acid.
After deprotection the peptide can then be elongated, to the desired
length using methods well known in the art to give (8). The coupling of
the protected amino acid subsequent to the building unit is performed by
such coupling agents exemplified by PyBrOP.RTM. to ensure high yield.
After the linear, resin bound peptide, e.g. (8), has been prepared the
.omega.-alkylene-protecting groups for example Boc and t-Bu are removed by
mild acid such as TFA to give (9). The resin bound peptide is then divided
into several parts. One part is subjected to on-resin cyclization using
for example TBTU as cyclization agent in DMF to ensure high yield of
cyclization, to give the N-backbone to side chain cyclic peptide resin
(10). After cyclization on the resin the terminal amino protecting group
is removed by agents such as piperidine and the backbone to side chain
cyclic peptide (11) is obtained after treatment with strong acid such as
HF. Alternatively, prior to the removal of the backbone cyclic peptide
from the resin, the terminal amino group is blocked by acylation with
agents such as acetic anhydride, benzoic anhydride or any other acid such
as adamantyl carboxylic acid activated by coupling agents such as BOP.
The other part of the peptide-resin (9) undergoes protecting of the side
chains used for cyclization, for example the .omega.-amino and carboxy
groups. This is done by reacting the .omega.-amino group with for example
Ac.sub.2 O and DMAP in DMF and activating the free .omega.-carboxy group
by for example DIC and HOBT to give the active ester which is then reacted
with for example Dh.sub.3 NH.sub.2 to give the linear analog (13) of the
cyclic peptide (10). Removal of the peptide from the resin and subsequent
removal of the side chains protecting groups by strong acid such as HF to
gives (14) which is the linear analog of the backbone to side chain cyclic
peptide (11).
The linear analogs are used as reference compounds for the biological
activity of their corresponding cyclic compounds.
[Reaction Scheme I follows at this point]
##STR37##
The selection of N.sup..alpha. and side chain protecting groups is, in
part, dictated by the cyclization reaction which is done on the
peptide-resin and by the procedure of removal of the peptide from the
resin. The N.sup..alpha. protecting groups are chosen in such a manner
that their removal will not effect the removal of the protecting groups of
the N.sup..alpha. (.omega.-aminoalkylene) protecting groups. In addition,
the removal of the N.sup..alpha. (.omega.-aminoalkylene)protecting groups
or any other protecting groups on .omega.-functional groups prior to the
cyclization, will not effect the other side chain protection and/or the
removal of the peptide from the resin. The selection of the side chain
protecting groups other than those used for cyclization is chosen in such
a manner that they can be removed subsequently with the removal of the
peptide from the resin. Protecting groups ordinarily employed include
those which are well known in the art, for example, urethane protecting
substituents such as Fmoc, Boc, Alloc, Z and the like.
It is preferred to utilize Fmoc for protecting the .alpha.-amino group of
the amino acid undergoing the coupling reaction at the carboxyl end of
said amino acid. The Fmoc protecting group is readily removed following
such coupling reaction and prior to the subsequent step by the mild action
of base such as piperidine in DMF. It is preferred to utilize Boc for
protecting .omega.-amino group of the N.sup..alpha.
(.omega.-aminoalkylene) group and t-Bu for protecting the carboxy group of
the amino acids undergoing the reaction of backbone cyclization. The Boc
and t-Bu protecting groups are readily removed simultaneously prior to the
cyclization.
(Scheme II)
Preparation of Peptides with Backbone to Backbone Cyclization
Preparation of N-backbone to N-backbone cyclized peptide analogs is
exemplified in scheme II. In this schematic example, the building group
consists of alkylene spacers and two amide bonds.
A building unit for backbone cyclization (the preparation of which
described in Schemes III-VIII) is coupled to a peptide resin, for example
peptide-resin (4), using for example the coupling reagent BOP to give
(16). One such building unit is for example
Fmoc-N.alpha.(.omega.-Boc-amino alkylene)amino acid. The side chain Boc
protecting group is removed by mild acid such as TFA in DCM and an N-Boc
protected .omega.-amino acid, or any other Boc protected amino acid, is
coupled to the side chain amino group using coupling agent such as BOP to
give peptide-resin (17).
After deprotection of the N.sup..alpha. Fmoc protecting group by mild base
such as piperidine in DMF, the peptide can then be elongated, if required,
to the desired length using methods well known in the art to give (18).
Alternatively, the deprotection of the N.sup..alpha. Fmoc and subsequent
elongation of the peptide can be done before deprotection of the side
chain Boc protecting group. The elongation of the N-alkylene side chain
allow control of the ring size. The coupling of the protected amino acid
subsequent to the building unit is performed by such coupling agents
exemplified by PyBrOP.RTM. to ensure high yield.
After deprotection of the terminal N.sup..alpha. Fmoc group, a second
building unit, for example Fmoc-N.sup..alpha.
(.omega.-t-Bu-carboxyalkylene)amino acid is coupled to the peptide-resin
to give (19). After deprotection of the N.sup..alpha. Fmoc protecting
group, the peptide can then be elongated, if required, to the desired
length using methods well known in the art to give (20). The coupling of
the protected amino acid subsequent to the building unit is performed by
such coupling agents exemplified by PyBrOP.RTM. to ensure high yield.
After the linear, resin bound peptide, e.g. (20), has been prepared the
.omega.-alkylene-protecting groups for example Boc and t-Bu are removed by
mild acid such as TFA to give (21). The resin peptide is then divided into
several parts. One part is subjected to on-resin cyclization using for
example TBTU as cyclization agent in DMF to ensure high yield of
cyclization, to give the N-backbone to N-backbone cyclic peptide resin
(22). After cyclization on the resin the terminal amino protecting group
is removed by agents such as piperidine and the backbone to backbone
cyclic peptide (23) is obtained after treatment with strong acid such as
HF. Alternatively, prior to the removal of the backbone cyclic peptide
from the resin, the terminal amino group of (22) is blocked, after
deprotection, by acylation with agents such as acetic anhydride, benzoic
anhydride or any other acid such as adamantyl carboxylic acid activated by
coupling agents such as BOP to give the N-terminal blocked backbone to
backbone cyclic peptide (24).
The other part of the peptide-resin (21) undergoes protecting of the side
chains used for cyclization, for example the .omega.-amino and carboxy
groups. This is done by reacting the .omega.-amino group with for example
Ac.sub.2 O and DMAP in DMF and activating the free .omega.-carboxy group
by for example DIC and HOBT to give the active ester which is then reacted
with for example MeNH.sub.2. Removal of the peptide from the resin and
subsequent removal of the side chains protecting groups by strong acid
such as HF to gives (26) which is the linear analog of the backbone to
backbone cyclic peptide (23). The linear analogs are used as reference
compounds for the biological activity of their corresponding cyclic
compounds.
Reaction Scheme II Follows at this Point
##STR38##
##STR39##
Novel Synthesis of Building Units
The novel synthesis providing N(.omega.-(functionalized) alkylene) amino
acids used to generate backbone cyclic peptides is depicted in schemes
III-VIII. In this approach we have implemented the following changes in
order to devise a practical, general synthesis:
1. The nucleophile is a secondary nitrogen, which is a better nucleophile
than the primary nitrogen previously used. This also prevents the
possibility of double alkylation.
2. The leaving group was changed to trifluoromethanesulfonyl (triflate),
which has a much lower tendency to eliminate than a halogen, thus making
it possible to implement the synthesis with amino acids other than
glycine. Furthermore, the triflate leaving group prevents racemization
during the alkylation reaction.
3. The carboxylate is esterified prior to the substitution reaction, to
facilitate the substitution by removing the negative charge next to the
electrophilic carbon.
(Scheme III)
Preparation of N.sup..alpha., N.sup..omega. Protected .omega.-amino
Alkylene Amino Acids Building Units
One preferred procedure for the preparation of protected N.sup..alpha.
(.omega.-amino alkylene) amino acids involves the N.sup..alpha. alkylation
of suitably protected diamino alkanes. One preferred
N.sup..alpha.,N.sup..omega. di- protected diamino alkane is for example
N.sup..alpha. -Benzyl, N.sup..omega. -Boc diamino alkane (27). This
starting material contains one protecting group such as Boc which is
necessary for the final product, and a temporary protecting group such as
Bzl to minimize unwanted side reactions during the preparation of the
titled compound. One preferred procedure for the preparation of the
starting material (27) involves reductive alkylation of N-Boc diamino
alkane with aldehydes such as benzaldehyde. The temporary protection of
the N.sup..alpha. amino group, which is alkylated in the reaction by such
protecting groups as Bzl, minimizes the dialkylation side reaction and
allows removal by such conditions that do not remove the N.sup..omega.
-protecting group.
The N.sup..alpha.,N.sup..omega. di-protected diamino alkane is reacted with
for example chiral .alpha.-hydroxy .alpha.-substituted acid esters where
the hydroxyl moiety is converted to a leaving group for example Triflate.
The use of Triflate as the leaving group was found to be superior to other
leaving groups such as halogens, Tosyl, Mesyl, etc., because it prevents
the .beta.-elimination reaction encountered with the other leaving groups.
The use of Triflate as the leaving group also ensures high optical purity
of the product (28). The temporary N.sup..alpha. protecting group, such as
Bzl, and the carboxyl protecting group, such as methyl ester, are removed
by mild conditions, such as catalytic hydrogenation and hydrolysis, that
do not remove the N.sup..omega. protecting group such as Boc to give the
N.sup..omega. protected amino acid (29). Introduction of the N.sup..alpha.
protecting group suitable for peptide synthesis is accomplished by methods
well known in the art, to give the protected N.sup..alpha. (N.sup..omega.
protected amino alkylene) amino acid (30).
The choice of the N.sup..alpha. and the N.sup..omega. protecting groups is
dictated by the use of the building units in peptide synthesis. The
protecting groups have to be orthogonal to each other and orthogonal to
the other side chains protecting groups in the peptide. Combinations of
N.sup..alpha. and N.sup..omega. protecting groups are for example:
N.sup..alpha. -Fmoc, N.sup..omega. -Boc; N.sup..alpha. -Fmoc,
N.sup..omega. -Alloc; N.sup..alpha. -Boc, N.sup..omega. -Alloc. These
combinations are suitable for peptide synthesis and backbone cyclization,
either on solid support or in solution.
(Scheme IV)
Preparation of N.sup..alpha., N.sup..omega. Protected .omega.-amino
Alkylene Glycine Building Units
One preferred procedure for the preparation of protected N.sup..alpha.
(.omega.-amino alkylene) glycines involves the reaction of the
N.sup..alpha., N.sup..omega. di-protected di amino alkane (27) with
commercially available .alpha.-activated carboxylic acid esters, for
example benzylbromo acetate. Since the titled compound is achiral, the use
of leaving groups such as Trf, Tos or Mes is not necessary. The use of the
same temporary protecting groups for the N.sup..alpha. and the carboxy
groups, for example the Bzl protecting group, ensures the prevention of
the undesired dialkylation side reaction and allows concomitant removal of
the temporary protecting groups thus giving high yield of the
N.sup..omega. protected amino acid (32). Introduction of the N.sup..alpha.
protecting group suitable for peptide synthesis is accomplished by methods
well known in the art, to give the protected N.sup..alpha. (N.sup..omega.
protected amino alkylene) glycines (33).
The choice of the N.sup..alpha. and the N.sup..omega. protecting groups is
dictated by the use of the building units in peptide synthesis. The
protecting groups have to be orthogonal to each other and orthogonal to
the other side chains protecting groups in the peptide. Combinations of
N.sup..alpha. and N.sup..omega. protecting groups are for example:
N.sup..alpha. Fmoc, N.sup..omega. Boc ; N.sup..alpha. Fmoc, N.sup..omega.
Alloc; N.sup..alpha. Boc, N.sup..omega. Alloc. These combinations are
suitable for peptide synthesis and backbone cyclization, either on solid
support or in solution.
##STR40##
##STR41##
##STR42##
(Scheme V)
Preparation of N.sup..alpha., .omega.-carboxy Protected .omega.-carboxy
Alkylene Amino Acids
One preferred procedure for the preparation of protected N.sup..alpha.
(.omega.-carboxy alkylene) amino acids involves the N.sup..alpha.
-alkylation of suitably N.sup..alpha., .omega.-carboxy deprotected amino
acids. One preferred deprotected amino acid is N.sup..alpha. -Benzyl
.omega.-amino acids t-butyl esters (34). This starting material contains
one protecting group such as t-Bu ester which is necessary for the final
product, and a temporary protecting group such as N.sup..alpha. Bzl to
minimize side reactions during the preparation of the titled compound. One
preferred procedure for the preparation of the starting material (34)
involves reductive alkylation of .omega.-amino acids t-butyl esters with
aldehydes such as benzaldehyde. The temporary protection of the amino
group which is used as nucleophile in the proceeding alkylation reaction
by such protecting groups as Bzl minimizes the dialkylation side reaction.
The N.sup..alpha., .omega.-carboxy deprotected amino acids (34) are reacted
with, for example, chiral .alpha.-hydroxy .alpha.-substituted acid esters
where the hydroxyl moiety is converted to a leaving group, for example,
Triflate. The use of Triflate as the leaving group was found to be
superior to other leaving groups such as halogens, Tosyl, Mesyl; etc.,
because it prevents the .beta.-elimination reaction encountered with the
other leaving groups. The use of Triflate as the leaving group also
ensures high optical purity of the product, for example (36). The
temporary N.sup..alpha. protecting group, such as Bzl, and the
.alpha.-carboxyl protecting group, such as benzyl ester, are concomitantly
removed by mild condition, such as catalytic hydrogenation, that to not
remove the .omega.-carboxy protecting group such as t-Bu to give the
N.sup..alpha. (protected .omega.-carboxy alkylene) amino acid (36).
Introduction of the N.sup..alpha. protecting group suitable for peptide
synthesis is accomplished by methods well known in the art, to give the
protected N.sup..alpha. (.omega. protected carboxy alkylene) amino acid
(37).
The choice of the N.sup..alpha. and the .omega.-carboxy protecting groups
is dictated by the use of the building units in peptide synthesis. The
protecting groups have to be orthogonal to each other and orthogonal to
the other side chains protecting groups in the peptide. A combination of
N.sup..alpha. and .omega.-carboxy protecting groups are for example:
N.sup..alpha. -Fmoc, .omega.-carboxy t-Bu; N.sup..alpha. -Fmoc,
.omega.-carboxy Alloc; N.sup..alpha. -Boc, .omega.-carboxy Alloc. These
combinations are suitable for peptide synthesis and backbone cyclization,
either on solid support or in solution.
(Scheme VI)
Preparation of N.sup..alpha., .omega.-carboxy Protected .omega.-carboxy
Alkylene Glycine Building Units
One preferred procedure for the preparation of protected N.sup..alpha.
(.omega.-carboxy alkylene)glycines involves the N.sup..alpha. -alkylation
of suitably N.sup..alpha., .omega.-carboxy deprotected amino acids (34)
with commercially available .alpha.-activated carboxylic acid esters for
example, benzyl bromo acetate. Since the titled compound is achiral, the
use of leaving groups such as Trf, Tos or Mes is not necessary.
The use of the same temporary protecting groups for the N.sup..alpha. and
the .alpha.-carboxy groups, for example the Bzl protecting group, ensures
the prevention of the undesired dialkylation side reaction and allows
concomitant removal of the temporary protecting groups thus giving high
yield of the N.sup..alpha. (protected .omega.-carboxy alkylene) glycines
(39). Introduction of the N.sup..alpha. protecting group suitable for
peptide synthesis is accomplished by methods well known in the art, to
give the protected N.sup..alpha. (.omega. protected carboxy alkylene)
glycines (40).
The choice of the N.sup..alpha. and the .omega.-carboxy protecting groups
is dictated by the use of the building units in peptide synthesis. The
protecting groups have to be orthogonal to each other and orthogonal to
the other side chains protecting groups in the peptide. A combination of
N.sup..alpha. and .omega.-carboxy protecting groups are, for example:
N.sup..alpha. Fmoc, .omega.-carboxy t-Bu; N.sup..alpha. Fmoc,
.omega.-carboxy Alloc; N.sup..alpha. Boc, .omega.-carboxy Alloc. These
combinations are suitable for peptide synthesis and backbone cyclization,
either on solid support or in solution.
##STR43##
##STR44##
(Scheme VII)
Preparation of N.sup..alpha. S.sup..omega. Protected .omega.-thio Alkylene
Amino Acid Building Units
One preferred procedure for the preparation of N.sup..alpha., S.sup..omega.
-deprotected N.sup..alpha. (.omega.-thio alkylene) amino acids involves
the N.sup..alpha. -alkylation of suitably S.sup..omega. protected
.omega.-thio amino alkanes. Suitable S.sup..omega. protecting groups are,
for example, Bzl, t-Bu, Trt. One preferred S.sup..omega. -protected
.omega.-thio amino alkanes is for example .omega.-(S-Benzyl) amino alkanes
(41). One preferred procedure for the preparation of the starting material
(41) involves the use of salts of S-protected thiols as nucleophiles for a
nucleophilic substitution reaction on suitably N.sup..alpha. -protected
.omega.-activated amino alkanes. Removal of the amino protection gives the
starting material (41).
The S-protected .omega.-thio amino alkanes (41) are reacted with for
example chiral .alpha.-hydroxy .alpha.-substituted acid esters where the
hydroxyl moiety is converted to a leaving group for example Triflate. The
use of Triflate as the leaving group was found to be superior to other
leaving groups such as halogens, Tosyl, Mesyl etc. because it prevents the
.beta.-elimination reaction encountered with the other leaving groups. The
use of Triflate as the leaving group also ensures high optical purity of
the product for example (42). The temporary .alpha.-carboxyl protecting
group, such as methyl ester, is removed by mild condition, such as
hydrolysis with base, that to not remove the .omega.-thio protecting group
such as S-Bzl to give the N.sup..alpha. (S-protected .omega.-thio
alkylene) amino acid (43). Introduction of the N.sup..alpha. protecting
group suitable for peptide synthesis is accomplished by methods well known
in the art, to give the protected N,S protected N.sup..alpha.
(.omega.-thio alkylene) amino acid (44).
The choice of the N.sup..alpha. and the .omega.-thio protecting groups is
dictated by the use of the building units in peptide synthesis. The
protecting groups have to be orthogonal to each other and orthogonal to
the other side chains protecting groups in the peptide. A combination of
N.sup..alpha. and .omega.-thio protecting groups are for example:
N.sup..alpha. Fmoc, S.sup..omega. t-Bu; N.sup..alpha. Fmoc, S.sup..omega.
Bzl; N.sup..alpha. Fmoc, S.sup..omega. Trt; N.sup..alpha. Boc,
S.sup..omega. Bzl. These combinations are suitable for peptide synthesis
and backbone cyclization, either on solid support or in solution.
(Scheme VIII)
Preparation of N.sup..alpha., S.sup..omega. Protected .omega.-thio Alkylene
Glycine Building Units
One preferred procedure for the preparation of N.sup..alpha., S.sup..omega.
-deprotected N.sup..alpha. (.omega.-thio alkylene) amino acids involves
the N.sup..alpha. -alkylation of suitably S.sup..omega. protected
.omega.-thio amino alkanes (41) with commercially available
.alpha.-activated carboxylic acid esters for example ethyl bromo acetate.
Since the titled compound is achiral, the use of leaving groups such as
Trf, Tos or Mes is not necessary.
Suitable protecting groups for the .omega.-thio groups are for example Bzl,
t-Bu, Trt. One preferred S-protected .omega.-thio amino alkanes is for
example .omega.-(S-Benzyl) amino alkanes (41). The N-alkylation reaction
gives the ester (45). The temporary .alpha.-carboxyl protecting group,
such as ethyl ester, is removed by mild conditions, such as hydrolysis
with base, that to not remove the .omega.-thio protecting group such as
S-Bzl to give the N.sup..alpha. (S-protected .omega.-thio alkylene)
glycines (46). Introduction of the N.sup..alpha. protecting group suitable
for peptide synthesis is accomplished by methods well known in the art, to
give the protected N.sup..alpha., S.sup..omega. -deprotected N.sup..alpha.
(.omega.-thio alkylene) glycines (47).
The choice of the N.sup..alpha. and the .omega.-thio protecting groups is
dictated by the use of the building units in peptide synthesis. The
protecting groups have to be orthogonal to each other and orthogonal to
the other side chains protecting groups in the peptide. A combination of
N.sup..alpha. and .omega.-thio protecting groups are for example:
N.sup..alpha. Fmoc, S.sup..omega. t-Bu; N.sup..alpha. Fmoc, S.sup..omega.
Bzl; N.sup..alpha. Fmoc, S.sup..omega. Trt; N.sup..alpha. Boc,
S.sup..omega. Bzl. These combinations are suitable for peptide synthesis
and backbone cyclization, either on solid support or in solution.
##STR45##
##STR46##
SPECIFIC EXAMPLES OF PEPTIDES
Preparation of the novel backbone cyclized peptide analogs using the
schematics outlined above will be illustrated by the following
non-limiting specific examples:
EXAMPLE 1
Ada-(D)Arg-Arg-cyclo(N.sup..alpha.
(1-(6-aminohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH
STAGE 1
Boc-Arg(Tos)-O-resin.fwdarw.Fmoc-Phe-Arg(Tos)-O-resin
Boc-L-Arg(Tos)-O-resin (0.256 g, 0.1 mmole, 0.39 meq of nitrogen/g) was
placed in a shaker flask and swelled for two hours by the addition of DCM.
The resin was then carried out through the procedure in Table 1 which
includes two deprotections of the Boc protecting group with 55% TFA in DCM
for a total of 22 minutes, washing, neutralization with 10% DIEA in NMP
and washing (Table 1 steps 1-8). After positive ninhydrin test, as
described in Kaiser et al., Anal Biochem., 34:595, 1970 and is
incorporated herein by reference in its entirety, coupling (Table 1 steps
9-10) was achieved in NMP by the addition of Fmoc-L-Phe (0.232 g, 0.6
mmole) and after 5 minutes of shaking, solid BOP reagent (0.265 g, 0.6
mmole) was added to the flask.
TABLE 1
PROCEDURE FOR 0.1 mMOLE SCALE
VOL- RE-
STEP SOLVENT/ UME TIME PEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT
1 DCM 5 120 1 Swells resin
2 DCM 5 2 3
3 TFA/DCM 55% 5 2 1 Deprotection
4 TFA/DCM 55% 5 20 1 Deprotection
5 DCM 5 2 3
6 NMP 5 2 4 check for
positive nin.
7 DIEA/NMP 5 5 2 Neutralization
8 NMP 5 2 5
9 Fmoc-AA in NMP 5 5 Coupling add
BOP 6 eq. add
DIEA 120 600 1
12 eq. Check
pH, adjust to
pH 8 with DIEA
10 NMP 5 2 5 check for
negative nin.
11 Pip/NMP 20% 5 10 1 Deprotection
12 Pip/NMP 20% 5 10 1
13 NMP 5 2 6 check for
positive nin.
After shaking for 10 minutes, the mixture was adjusted to pH 8 (measured
with wetted pH stick) by the addition of DIEA (0.209 mL, 1.2 mmole) and
the flask shaken for 10 hours at ambient temperature. The resin was then
washed and subjected to ninhydrin test. After negative ninhydrin test the
resin was used for the next coupling.
STAGE 2
Fmoc-Phe-Arg(Tos)-O-resin.fwdarw.Fmoc-N.sup..alpha. (6-Boc amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin
The Fmoc-Phe-Arg(Tos)-O-resin (Stage 1) was subjected to two deprotections
of the Fmoc protecting group by 20% Pip in NMP (Table 1 steps 11-13).
After washing and ninhydrin test (Method J, below), coupling of Fmoc-D-Phe
was achieved as described in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Phe
(0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL,
1.2 mmole). The resin was washed and the Fmoc group deprotected as
described above (Table 1 steps 11-13) After washing and ninhydrin test,
coupling of Fmoc-D-Asp(t-Bu) was achieved as described in Stage 1 (Table 1
steps 9-10) using Fmoc-D-Asp(t-Bu) (0.247 g, 0.6 mmole), BOP reagent
(0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed
and the Fmoc group deprotected as described above (Table 1 steps 11-13).
After washing and ninhydrin test, coupling of Fmoc-L-Phe was achieved as
described in Stage 1 (Table 1 steps 9-10) using Fmoc-L-Phe (0.232 g, 0.6
mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).
The resin was washed and the Fmoc group deprotected as described above
(Table 1 steps 11-13). After washing and ninhydrin test, coupling of
Fmoc-L-Hyp(OBzl) was achieved as described in Stage 1 (Table 1 steps 9-10)
using Fmoc-L-Hyp(OBzl) (0.266 g, 0.6 mmole), BOP reagent (0.265 g, 0.6
mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and the Fmoc
group deprotected as described above (Table 1 steps 11-13). The resin was
washed and subjected to picric acid test (Method K). Coupling of
Fmoc-N.sup..alpha. (6-Boc amino hexylene) glycine was achieved as
described in Stage 1 (Table 1 steps 9-10) using Fmoc-N.sup..alpha. (6-Boc
amino hexylene)glycine (0.3 g, 0.6 mmole), BOP reagent (0.265 g, 0.6
mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was then washed and
subjected to the picric acid test (Method K below). After negative test
the resin was used for the next coupling.
STAGE 3
Fmoc-N.sup..alpha. (6-Boc amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin.fwdarw.F
moc-D-Arg(Tos)-Ara(Tos)-N.sup..alpha. (6-Boc amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin
The Fmoc-N.sup..alpha. (6-Boc amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage
2) was subjected to three deprotection of the Fmoc protecting group by 20%
Pip in NMP (Table 2 steps 1-2). After washing, the picric acid test
(Method K) was performed. If the test did not show 98.+-.2%, deprotection
of the peptide resin was subjected again to 3 deprotection steps (Table 2
steps 1-2), washing and picric acid test (Method K). Coupling of
Fmoc-L-Arg(Tos) was achieved in NMP by the addition of (0.33 g, 0.6 mmole)
and after 5 minutes of shaking, solid PYBOP reagent (0.28 g, 0.6 mmole)
was added to the flask. After shaking for 10 minutes, the mixture was
adjusted to pH 8 (measured with wetted pH stick) by the addition of DIEA
(0.209 mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient
temperature. The resin was then washed and subjected to a second coupling
by the same procedure for 20 hours. After washing the resin was subjected
to picric acid test (Method K) (Table 2 steps 3-6). If the test did not
show 98.+-.2% coupling the peptide resin was subjected again to a third
coupling for 2 hours at 50.degree. C. (Table 2 step 7). The resin was
washed subjected to three deprotection of the Fmoc protecting group by 20%
Pip in NMP (Table 2 steps 1-2). After washing picric acid test (Method K)
was performed.
TABLE 2
PROCEDURE FOR 0.1 mMOLE SCALE
VOL- RE-
STEP SOLVENT/ UME TIME PEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT
1 Piperidine/NMP 5 10 3 Deprotection
20%
2 NMP 5 2 6 Picric acid test.
3 Fmoc-AA in NMP 5 5 Coupling
add PyBroP 6 eq.
add DIEA 150 1 12 eq. Check pH,
adjust to pH 8
with DIEA.
4 NMP 5 2 3 check for
negative
5 Fmoc-AA in NMP 5 5 Coupling
add PyBroP 6 eq.
add DIEA 20 hr. 1 12 eq.
Check pH, adjust
to pH 8 with
DIEA.
6 NMP 5 2 4 Picric acid test.
If less than
98 .+-. 2%
coupling repeat
Steps 4-5
7 Fmoc-AA in NMP 5 5 Coupling at
add PyBOP 50.degree. C. 6 eq.
add DIEA 120 1 12 eq.
Check pH, adjust
to pH 8 with
DIEA.
8 NMP 5 2 4
If the test did not show 98.+-.2% deprotection, the peptide resin was
subjected again to 3 deprotection steps (Table 2 steps 1-2), washing and
the picric acid test (Method K). Coupling of Fmoc-D-Arg(Tos) was achieved
in NMP as described in Stage 1 (Table 1 steps 9-10) using Fmoc-D-Arg(Tos)
(0.33 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL,
1.2 mmole). The resin was washed 6 times with NMP (Table 1 step 15) and
used in the next stages.
STAGE 4
Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup..alpha. (6-Boc amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin.fwdarw.A
da-D-Arg-Arg-cyclo(N.sup..alpha.
(1-(6-amidohexylene)Gly-Hyp-Phe-D-Asp)-D-Phe-Phe-Arg-OH
The Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup..alpha. (6-Boc amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp(t-Bu)-D-Phe-Phe-Arg(Tos)-O-resin (Stage
3) was subjected to deprotection of the Boc and t-Bu protecting groups and
on resin cyclization according to Table 3. The peptide resin was washed
with DCM and deprotected as described in Stage 1 by 55% TFA in DCM. After
washing and neutralization by 10% DIEA in NMP and washing 6 times with DCM
the peptide resin was dried in vacuo for 24 hours. The dry peptide resin
weight, 0.4 g, it was divided into two parts. 0.2 g of the peptide resin
was swollen 2 hours in 5 mL NMP and cyclized as follows: Solid TBTU
reagent (0.19 g, 6 mmole) was added to the flask. After shaking for 10
minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209
mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature.
The resin was then washed and subjected to a second coupling by the same
procedure for 20 hours. After washing the resin was subjected to picric
acid test (Method K) (Table 3 steps 8-11). If the test did not show
98.+-.2% cyclization the peptide resin was subjected again to a third
cyclization for 2 hours at 50.degree. C. (Table 2 step 12). The resin was
washed, subjected to three deprotection of the Fmoc protecting group by
20% Pip in NMP (Table 2 steps 1-2). After washing and ninhydrin test, the
N-terminal amino group was blocked by Ada. Adamantane acetic acid (0.108
g, 6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2
mmole) were added and the flask shaken for 2 hours. After washing 6 times
with NMP (Table 2 step 13), ninhydrin test (Method J) was performed. If
the test was positive or slightly positive the protecting with adamantane
acetic acid was repeated. If the ninhydrin test was negative, the peptide
resin was washed 6 times with NMP and 6 times with DCM. The resin was
dried under vacuum for 24 hours. The dried resin was subjected to HF as
follows: to the dry peptide resin (0.2 g) in the HF reaction flask,
anisole (2 mL) was added and the peptide treated with 20 mL liquid HF at
-20.degree. C. for 2 hours. After the evaporation of the HF under vacuum,
the anisole was washed with ether (20 mL, 5 times) and the solid residue
dried in vacuum. The peptide was extracted from the resin with TFA (10 mL,
3 times) and the TFA evaporated under vacuum. The residue was dissolved in
20 mL 30% AcOH and lyophilized. This process was repeated 3 times. The
crude peptide was purified by semiprep HPLC (Method H). The final product
was obtained as white powder by lyophilization from dioxane, which gave 42
mg (56%) of the title compound.
HPLC (Method G) RT 32.15 minutes, 95%
TOF MS: 1351.4 (M.sup.+)
AAA in agreement with the title compound
TABLE 3
PROCEDURE FOR 0.05 mMOLE SCALE
VOL- RE-
STEP SOLVENT/ UME TIME PEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT
1 DCM 5 2 3
2 TFA/DCM 55% 5 2 1 Deprotection
3 TFA/DCM 55% 5 20 1 Deprotection
4 DCM 5 2 3
5 NMP 5 2 4
6 DIEA/NMP 10% 5 5 2 Neutralization
7 NMP 5 2 5
8 TBTU/NMP/ 5 150 3 Cyclization
DIEA
9 NMP 5 2 4 Picric acid test.
If less than 98 .+-. 2%
coupling perform
Steps 10-12. If
above 98 .+-. 2%, go
to step 13.
10 TBTU/NNP/ 5 20 hr 3 Cyclization. Check
DIEA pH, adjust to pH 8
with DIEA.
11 NMP 5 2 4 Picric acid test.
If less than 98 .+-. 2%
coupling perform
Steps 12. If above
98 .+-. 2%, go to
step 13
12 TBTU/NMP/ 5 120 3 Cyclization, 50 C.
DIEA Check pH, adjust to
pH 8 with DIEA.
13 NMP 5 2 6
14 Pip/NMP 20% 5 10 1 Deprotection
15 Pip/NMP 20% 5 10 1
16 NMP 5 2 6 Check for positive
nin.
17 AdacOH/BOP/ 5 2 1
NMP
18 NMP 5 2 6 Check for negative
nin.
19 DCM 5 2 4
EXAMPLE 2
NON-CYCLIZED PEPTIDE (Control for biological assays)
Ada-D-Arg-Arg-N.sup..alpha.
(6-acetamidohexylene)Gly-Hyp-Phe-D-Asp(NH-Me)-D-Phe-Phe-Arg-OH
The Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup..alpha. (6-amino
hexylene)Gly-Hyp(OBzl)-Phe-D-Asp-D-Phe-Phe-Arg(Tos)-O-resin (0.2 g) which
was prepared in Example 1 Stage 4 was subjected to acetylation of the
6-amino side chain of N.sup..alpha. (6-acetamidohexylene)Gly and to methyl
amidation of the carboxylic group of D-Asp as described in Table 4. The
peptide resin was swollen in 5 mL NMP for 2 hours and AcO (0.113 mL, 12
mmole) and PP (17 mg) were added. After 30 minutes, the resin was washed
with NMP 6 times and subjected to ninhydrin test. If the test was positive
or slightly positive the acetylation reaction was repeated. If the
ninhydrin test was negative, the carboxy group of D-Asp was activated by
the addition of HOBT (0.040 g, 0.3 mmole) and DIC (0.047 mL, 0.3 mmole) to
the peptide resin in NMP. The mixture was shaken for half an hour and a
solution of 30% methylamine in EtOH (0.2 mL) was added. After one hour,
the resin was washed 6 times with NMP and the terminal Fmoc group removed
by 20% Pip in NMP (Table 4 steps 7-9). After washing with NMP the
N-terminal amino group was blocked by Ada as described in Example 1 Stage
4 and the resin was washed with NMP and DCM (Table 4 steps 10-12) and the
resin dried in vacuo. The peptide was deprotected and cleaved from the
resin by HF. To the dry peptide resin (0.2 g) in the HF reaction flask,
anisole (2 mL) was added and the peptide treated with 20 mL liquid HF at
-20.degree. C. for 2 hours. After the evaporation of the HF under vacuum,
the anisole was washed with ether (20 mL 5 times) and the solid residue
dried in vacuo. The peptide was extracted from the resin with TFA (10 mL,
3 times) and the TFA evaporated under vacuum. The residue was dissolved in
20 mL 30% AcOH and lyophilized. This process was repeated 3 times. The
crude peptide was purified by semiprep HPLC (Method H). The final product
was obtained as white powder by lyophilization from dioxane, which gave 48
mg (64%) of the title compound.
HPLC (Method G) RT 27.70 minutes, 93%
TOF MS: 1424.6 (M.sup.+)
AAA in agreement with the title compound
TABLE 4
PROCEDURE FOR 0.05 mMOLE SCALE
VOL- RE-
STEP SOLVENT/ UME TIME PEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT
1 NMP 5 120 1 Swells resin
2 Ac.sub.2 O/PP/NMP 5 30 1 Protecting of side
chain
3 NMP 5 2 6 check for
negative nin.
4 DIC/HOBT/NMP 5 30 1 Activation of
COOH side chain
5 MeNH.sub.2 /EtOH/ 5 60 1 Protecting of side
chain
6 NMP 5 2 6
7 Pip/NMP 20% 5 10 1 Deprotection
8 Pip/NMP 20% 5 10 1
9 NMP 5 2 6 Check for
positive nin.
10 AdacOH/BOP/ 5 2 1
NMP
11 NMP 5 2 6 Check for
negative nin.
12 DCM 5 2 4
EXAMPLE 3
H-D-Arg-Arg-cyclo(N.sup..alpha. (1-(4-propanoyl))Gly-Hyp-Phe-N.sup..alpha.
(3-amido-propylene)Gly)-Ser-D-Phe-Phe-Arg-OH
STAGE 1
Fmoc-Phe-Arg(Tos)-O-resin.fwdarw.Fmoc-N.sup..alpha.
(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup..alpha. (3-Boc amino
propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin
Fmoc-Phe-Arg(Tos)-O-resin prepared from Boc-Arg(Tos)-O-Resin (0.3 g, 0.1
mmole) (Example 1, Stage 1) was subjected to two deprotection of the Fmoc
protecting group by 20% piperidine in NMP (Table 1, steps 11-13). After
washing and ninhydrin test (Method J), coupling of Fmoc-D-Phe was achieved
as described in Stage 1 (Example 1) (Table 1 steps 9-10) using Fmoc-D-Phe
(0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL,
1.2 mmole). The resin was washed and the Fmoc group deprotected as
described above (Table 1, steps 11-13). After washing and ninhydrin test
(Method J), coupling of Fmoc-Ser(BzL) was achieved as described in Stage 1
(Example 1) (Table 1 steps 9-10) using Fmoc-Ser(Bzl) (0.25 g, 0.6 mmole),
BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin
was washed and the Fmoc group deprotected as described above (Table 1,
steps 11-13). After washing and picric acid test (Method K), coupling of
Fmoc-N.sup..alpha. (3-Boc amino propylene)glycine was achieved as
described in Table 1, steps 9-10 using Fmoc-N.sup..alpha. (3-Boc amino
propylene)Gly (0.272 g, 0.6 mmole), BOP reagent (0.265 g, 0.6 mmole) and
DIEA (0.209 mL, 1.2 mmole). The resin was washed and subjected to three
deprotection of the Fmoc protecting group by 20% Pip in NMP (Table 2,
steps 1-2). After washing picric acid test (Method K) was performed. If
the test did not show 98.+-.2% deprotection the peptide resin was
subjected again to 3 deprotection steps (Table 2, steps 1-2), washing and
picric acid test (Method K). Coupling of Fmoc-L-Hyp(OBzl) was achieved in
NMP by the addition of Fmoc-L-Hyp(OBzl) (0.33 g, 0.6 mmole) and after 5
minutes of shaking, solid PyBrOP reagent (0.28 g, 0.6 mmole) was added to
the flask. After shaking for 10 minutes, the mixture was adjusted to pH 8
by the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5
hours at ambient temperature. The resin was then washed and subjected to a
second coupling by the same procedure for 20 hours. After washing the
resin was subjected to picric acid test (Method K) (Table 2, steps 3-6).
If the test did not show 98.+-.2% coupling the peptide resin was subjected
again to a third coupling for 2 hours at 50.degree. C. (Table 2, step 7).
The resin was washed subjected to three deprotection of the Fmoc
protecting group by 20% Pip in NMP (Table 2, steps 1-2). After washing
picric acid test (Method K) was performed. If the picric acid test did not
show 98.+-.2% deprotection, the resin was subjected again to deprotections
steps (Table 2, steps 1-2). Coupling of Fmoc-Phe was achieved in NMP by
the addition of Fmoc-Phe (0.232 g, 0.6 mmole), BOP reagent (0.265 g, 0.6
mmole) and DIEA (0.209 mL, 1.2 mmole). The resin was washed and after
picric acid test (Method K) the Fmoc group deprotected as described above
(Table 2, steps 1-2). After washing picric acid test (Method K) was
performed. If the test did not show 98.+-.2% deprotection the peptide
resin was subjected again to 3 deprotection steps (Table 2, steps 1-2),
washing and picric acid test (Method K). Coupling of N.sup..alpha. (3-t-Bu
carboxy propylene)Gly was achieved as described in Table 1 steps 9-10
using N.sup..alpha. (3-t-Bu carboxy propylene)Gly (0.264 g, 0.6 mmole),
BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole). The resin
was then washed and subjected to the picric acid test (Method K). After
negative test the resin was used for the next coupling.
STAGE 2
Fmoc-N.sup..alpha. (4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup..alpha. (3-Boc
amino
propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin.fwdarw.Fmoc-D-Arg(Tos)-
Arg(Tos)-N.sup..alpha. (4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup..alpha.
(3-Boc amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin
Fmoc-N.sup..alpha. (4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup..alpha. (3-Boc
amino propylene)-Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 1) was
subjected to three deprotection of the Fmoc protecting group by 20%
Piperidine in NMP (Table 2, steps 1-2). After washing picric acid (Method
K) test was performed. If the test did not show 98.+-.2% deprotection the
peptide resin was subjected again to 3 deprotection steps (Table 6, steps
1-2), washing and picric acid test. Coupling of Fmoc-L-Arg(Tos) was
achieved in NMP by the addition of (0.33 g, 0.6 mmole) and after 5 minutes
of shaking, solid PyBroP reagent (0.28 g, 0.6 mmole) was added to the
flask. After shaking for 10 minutes, the mixture was adjusted to pH 8 by
the addition of DIEA (0.209 mL, 1.2 mmole) and the flask shaken for 2.5
hours at ambient temperature. The resin was then washed and subjected to a
second coupling by the same procedure for 20 hours. After washing the
resin was subjected to picric acid test (Method K) (Table 2, steps 3-6).
If the test did not show 98.+-.2% coupling the peptide resin was subjected
again to a third coupling for 2 hours at 50.degree. C. (Table 2, step 7).
The resin was washed subjected to three deprotection of the Fmoc
protecting group by 20% Pip in NMP (Table 2, steps 1-2). After washing
picric acid test (Method K) was performed. If the test did not show
98.+-.2% deprotection the peptide resin was subjected again to 3
deprotection steps (Table 2, steps 1-2), washing and picric acid test
(Method K). Coupling of Fmoc-D-Arg(Tos) was achieved in NMP as described
in Stage 1 (Table 1, steps 9-10) using Fmoc-D-Arg(Tos) (0.33 g, 0.6
mmole), BOP reagent (0.265 g, 0.6 mmole) and DIEA (0.209 mL, 1.2 mmole).
The resin was washed 6 times with NMP (Table 1, step 15) and used in the
next stages.
STAGE 3
Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup..alpha.
(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup..alpha. (3-Boc
amino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin.fwdarw.H-D-Arg-Arg
-cyclo(N.sup..alpha. (4-propanoyl))Gly-Hyp-Phe-N.sup..alpha.
(3-amido-propyl)Gly)-Ser-D-Phe-Phe-Arg-OH
Fmoc-D-Arg(Tos)-Arg(Tos)-N.sup..alpha.
(4-t-Bu-propanoyl)Gly-Hyp(OBzl)-Phe-N.sup..alpha. (3-Boc
amino-propylene)Gly-Ser(Bzl)-D-Phe-Phe-Arg(Tos)-O-resin (Stage 2) was
subjected to deprotection of the Boc and t-Bu protecting groups and on
resin cyclization according to Table 5. The peptide resin was washed with
DCM and deprotected as described in Stage 1 by 55% TFA in DCM. After
washing and neutralization by 10% DIEA in NMP and washing 6 times with and
NMP (Table 5, steps 1-5) the peptide was cyclized as follow: solid TBTU
reagent (0.19 g, 6 mmole) was added to the flask. After shaking for 10
minutes, the mixture was adjusted to pH 8 by the addition of DIEA (0.209
mL, 1.2 mmole) and the flask shaken for 2.5 hours at ambient temperature.
The resin was then washed and subjected to a second coupling by the same
procedure for 20 hours. After washing the resin was subjected to picric
acid test (Method K) (Table 3, steps 8-11). If the test did not show
98.+-.2% cyclization the peptide resin was subjected again to a third
cyclization for 2 hours at 50.degree. C. (Table 2, step 12). The resin was
washed, subjected to three deprotection of the Fmoc protecting group by
20% Pip in NMP (Table 5, steps 14-15). After washing 6 times with NMP and
4 times with DCM, the resin was dried in vacuo for 24 hours. The dried
resin was subjected to HF as follows: to the dry peptide resin (0.4 g) in
the HF reaction flask, anisole (2 mL) was added and the peptide treated
with 20 mL liquid HF at -20.degree. C. for 2 hours. After the evaporation
of the HF under vacuum, the anisole was washed with ether (20 mL, 5 times)
and the solid residue dried in vacuo. The peptide was extracted from the
resin with TFA (10 mL 3 times) and the TFA evaporated under vacuum. The
residue was dissolved in 20 mL 30% AcOH and lyophilized. This process was
repeated 3 times. The crude peptide was purified by semipreparative HPLC
(Method H). The final product was obtained as white powder by
lyophilization from dioxane, which gave 59 mg (34%) of the title compound.
[Table 5 follows at this point]
TABLE 5
PROCEDURE FOR 0.1 mMOLE SCALE
STEP SOLVENT/ VOLUME TIME REPEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT
1 DCM 10 2 3
2 TFA/DCM 10 2 1 Deprotection
55%
3 TFA/DCM 10 20 1 Deprotection
55%
4 DCM 10 2 3
5 NMP 10 2 4
6 DIEA/NMP 10 5 2 Neutralization
10%
7 NMP 10 2 5
8 TBTU/NMP/ 10 150 3 Cyclization
DIEA
9 NMP 10 2 4 Picric acid test.
If less than
98 .+-. 2%
Coupling
perform Steps 10.
10 TBTU/NMP/ 10 20 h 3 Cyclization,
DIEA Check pH, adjust
to pH 8 with
DIEA.
11 NMP 10 2 4 Picric acid test.
If less than
98 .+-. 2% coupling
perform Step 12.
12 TBTU/NMP/ 10 120 3 Cyclization, 50.degree.
DIEA C. Check pH,
adjust to pH 8
with DIEA.
13 NMP 10 2 6
14 Piper- 10 10 1 Deprotection
idine/
NMP 20%
15 Piper- 10 1
idine/
NMP 20%
16 NMP 10 2 6 Check for
positive nin.
17 DCM 10 2 4
HPLC (Method G)RT 33.62 minutes (91%)
TOF MS: 1278 (M.sup.+)
AAA in agreement with the title compound
EXAMPLE 4
H-D-Arg-Arg-cyclo(N.sup..alpha. (4-propanoyl)Gly-Hyp-Phe-N.sup..alpha.
(3-amido-propyl)-S-Phe)-Ser-D-Phe-Phe-Arg-OH
Title compound was synthesized according to Example 3 except that in stage
1, Fmoc-N.sup..alpha. (3-Boc-amino-propylene)-S-Phe (0.326) was
substituted for Fmoc-N.sup..alpha. (3-Boc amino propylene)Gly. A total of
0.643 g Boc-L-Arg(Tos)-O-resin (0.39 meq/g, 0.250 mmole) was used and
reagent quantities were adjusted accordingly. Cyclic peptide yield (from
half the total resin used) was 74 mg (42%) of the title compound.
SPECIFIC EXAMPLES OF BUILDING UNITS
The following specific examples of novel building units are provided for
illustrative purposes not meant to be limiting. The following is described
in sections, including "Procedures", "Methods", "Compounds" and
"EXAMPLES". "Procedures" are detailed stepwise descriptions of synthetic
procedures according to the more general schemes. "Methods" are general
descriptions of analyses used to determine the progress of the synthetic
process. Numbered "Compounds" are either the starting material or
intermediates for further numbered "Compounds" the synthesis of which
progresses according to the specified "Procedure." Several "Compounds"
used in series produce "EXAMPLES" of novel building units of the present
invention. For instance, "Compounds" 27-29, 41-44, 46-47, 51-55, 62, 63,
67 and 76-79 are actually "EXAMPLES" 5-25, respectively, of novel building
units of the present invention. "Compounds" 1-26, 30-40, 45, 48-50, 56-61,
64-66, 68-75 are starting materials or intermediates (only) for the
synthesis of "EXAMPLES".
Procedure 1
Synthesis of N-Boc alkylene diamines (BocNH(CH.sub.2).sub.n NH.sub.2)
(known compounds).
To a solution of 0.5 mole alkylene diamine in 0.5 L CHCl.sub.3 cooled in an
ice-water bath, was added dropwise, with stirring, a solution of 10.91 g
(0.05 mole) Boc.sub.2 O in 0.25 L CHCl.sub.3 for 3 h. The reaction mixture
was stirred for 16 h. at room temperature and then washed with water
(8.times.250 mL). The organic phase was dried over Na.sub.2 SO.sub.4 and
evaporated to dryness in vacuo.
Procedure 2
Synthesis of N-Boc, N-Bzl alkylene diamines (BocNH(CH.sub.2).sub.n NH-Bzl).
To a solution of 0.05 mole of mono Boc alkylene diamine in 60 mL MeOH was
added 2.77 mL (0.02 mole) Et.sub.3 N, 9.02 g (0.075 mole) MgSO.sub.4, and
5.56 mL (0.055 mole) of freshly distilled benzaldehyde. The reaction
mixture was stirred under room temperature for 1.5 h. Then 11.34 g (0.3
mole) of NaBH.sub.4 were added in small portions during 0.5 h with cooling
to -5.degree. C. The reaction mixture was then stirred for 1 h at
-5.degree. C. and for another 1 h at 0.degree. C. The reaction was stopped
by addition of 200 mL water and the product was extracted with EtOAc
(3.times.200 mL). The combined EtOAc extracts were washed with water
(4.times.100 mL). The organic phase was extract with 0.5 N HCl
(4.times.100 mL) and the aqueous solution was neutralized under cooling
with 25 mL 25% NH.sub.4 OH, extracted with CHCl.sub.3 (3.times.100 ml) and
the combined extracts were washed with water (3.times.80 mL), dried over
Na.sub.2 SO.sub.4 and evaporated to dryness in vacuo.
Procedure 3
Synthesis of (R) or (S) .alpha.-hydroxy acids (known compounds).
To a solution of 16.52 g (0.1 mole) (R) or (S) amino acid in 150 ml 1N
H.sub.2 SO.sub.4 was added dropwise a solution of 10.35 g (0.15 mole)
NaNO.sub.2 in 100 mL H.sub.2 O during 0.5 h with stirring and cooling in
an ice bath. The reaction mixture was stirred 3 h. at 0.degree. C. and
additional 18 h at room temperature, then the (R)- or (S)- hydroxy acid
was extracted with ether in a continuous ether extractor. The etheral
solution was washed with 1N HCl (2.times.50 mL), H.sub.2 O (3.times.80
mL), dried over Na.sub.2 SO.sub.4 and evaporated to dryness. The product
was triturated twice from ether:petrol-ether (40-60.degree. C.) (1:10).
The precipitate was filtered, washed with 50 mL petrol-ether and dried.
Procedure 4
Synthesis of (R) or (S) .alpha.-hydroxy acid methyl esters (known
compounds).
To a suspension of 0.065 mole (R)- or (S)- hydroxy acid in 100 mL ether was
added under cooling in an ice bath 300 mL of an etheral solution of
CH.sub.2 N.sub.2 until stable yellow color of reaction mixture was
obtained. Then the ether solution was washed with 5% KHCO.sub.3
(3.times.100 mL) and H.sub.2 O (2.times.80 mL), dried over Na.sub.2
SO.sub.4 and evaporated to dryness. The product was dried in vacuo.
Procedure 5
Synthesis of triflate of (R) or (S) .alpha.-hydroxy acid methyl esters.
To a cooled solution of 2.67 ml (0.033 mole) pyridine in 20 mL dry DCM was
added 5.55 mL (0.033 mole) Trf.sub.2 O at -20.degree. C. (dry ice in EtOH
bath), then after 5 min a solution of 0.03 mole (R) or (S) .alpha.-hydroxy
acid methyl ester in 20 mL dry DCM was added dropwise. The reaction
mixture was stirred at room temperature for 45 min, then was passed
through a short silica gel column (2 cm). The product was eluted with 400
mL of petrol-ether:methylene-chloride (1:1). The solvent was evaporated in
vacuo.
Procedure 6
Synthesis of (R) or (S) N.sup..alpha. (Bzl)(N.sup..omega. -Boc-amino
alkylene)amino acid methyl esters ((R) or (S) BocNH(CH.sub.2).sub.n
N(Bzl)CH(R)COOMe).
To a solution of 0.022 mole of N.sup..alpha. -Boc, N.sup..omega. -Bzl
alkylene diamine in 20 mL of dry DCM was added 3.04 mL (0.022 mole)
Et.sub.3 N. Then a solution of 0.02 mole of (R) or (S) .alpha.-hydroxy
acid methyl ester triflate in 25 mL dry DCM was added dropwise (0.5 h.)
under cooling in an ice-water bath. The reaction mixture was stirred at
room temperature for 18 h. Then 150 mL of CHCl.sub.3 was added and the
yellow solution was washed with water (3.times.80 mL). The organic phase
was dried over Na.sub.2 SO.sub.4 and adsorbed on silica-gel and dried in
vacuo. The silica-gel was washed on filter with 0.5 L of petrol-ether and
with 0.5 L of 2% EA in PE. Then the product was eluted from silica with
0.5 L of mixture petrol-ether:ethyl-acetate (4:1). The solvent was
evaporated in vacuo. If the product was not clean it was further purified
on a small column of silica-gel (250 mL). The first impurities were eluted
with 0.8 L of hexane then the product was eluted with 1.5 L of mixture of
petrol-ether:ethyl-acetate (4:1).
Procedure 7
Hydrolysis of methyl esters
To a solution of 0.015 mole of methyl ester in 40 mL MeOH was added 10 mL
7.5 N NaOH cooled in an ice-water bath. The reaction mixture was stirred
at room temperature for approximately 24 h (until the methyl ester spot
disappears on TLC). Then 100 mL of water were added and the reaction
mixture was washed with petrol-ether (3.times.80 mL). The aqueous solution
was acidified under cooling by addition of 40 mL 2N HCL. The product was
extracted with a mixture of CHCl.sub.3 :i-PrOH (3:1) (3.times.80 mL),
dried over Na.sub.2 SO.sub.4, evaporated to dryness and dried in vacuo to
obtain a white foam in quantitative yield.
Procedure 8
Removal of Bzl by Hydrogenation with Pd/C
To a solution of 0.012 mole (R) or (S) N.sup..alpha. (Bzl)(N.sup..omega.
-Boc-amino alkylene) amino acid in 60 mL MeOH--DMF (11-1) was added 0.5 g
10% Pd/C. The solution was hydrogenated for 4 h under a pressure of 45-50
Psi at room temperature. Then 200 mL of a mixture of DMF:MeOH:H.sub.2
O:glacial AcOH (1:3:5:1) was added. The catalyst was filtrated off and
washed (is the acetic acid?) with H.sub.2 O or MeOH (2.times.15 mL). The
combined filtrate was evaporated to dryness and recrystallized from
methanol:ether (15 mL:250). The precipitate was filtered and dried in
vacuo.
Procedure 9
Synthesis of (R) or (S) N.sup..alpha. (Fmoc)(N.sup..omega. -Boc-amino
alkylene) amino acid.
To 50 mL water was added 0.07 mole of (R) or (S) N.sup..alpha.
(N.sup..omega. -Boc-amino alkylene) amino acid and 1.95 mL (0.014 mole)
Et.sub.3 N. The suspension was stirred 2-3 h until a clear solution was
obtained. Then a solution of 2.25 g (0.07 mole) of FmocOSu in 100 mL ACN
was added. The reaction mixture was stirred 18 h at room temperature, then
150 mL water was added and the solution was washed with petrol-ether
(3.times.100 mL) and with ether:petrol-ether (1:4). The aqueous solution
was acidified by addition of 14 mL 1N HCL. The product was extracted with
EtOAc (4.times.100 mL) and the organic phase was washed with 0.5 N HCl
(2.times.50 mL), H.sub.2 O (3.times.80 mL), dried over Na.sub.2 SO.sub.4,
evaporated to dryness and recrystallized from ether:petrol-ether (80
mL:200 mL)
Procedure 10
Synthesis of S-benzylcysteamine (Bzl-S--(CH.sub.2).sub.2 --NH.sub.2) (known
compound).
To a suspension of 0.1 mole cysteamine hydrochloride in 20 mL methanol were
added 13.6 mL of 25% ammonia solution, followed by dropwise addition of
0.12 mole benzyl bromide at room temperature. The mixture was stirred for
0.5 h, and the formed precipitate of S-dibenzylcysteamine was collected by
filtration. The product was extracted with ether (3.times.100 mL) and the
organic phase was successively washed with brine (2.times.100 mL), dried
over MgSO.sub.4 and the solvent evaporated in vacuo. The crude product was
essentially pure enough for the next step. It could, however be
recrystallized from ethyl acetate. Yield 86%, of white solid. m.p.
85-6.degree. C. NMR (CDCl.sub.3) in agreement with the title compound.
Procedure 11
Synthesis of N.sup..alpha. -(.omega.-(benzylthio)alkylene)phthalimides
((Bzl-S--(CH.sub.2).sub.n --N.dbd.Pht)) (known compounds)
N-(.omega.-bromoalkylene)phthalimide (0.1 mole) and benzyl mercaptane (0.11
mole) were stirred with 0.1 mole potassium carbonate in 100 mL DMSO at
50.degree. C. for 24 hours. The mixture was poured into ice-water and the
product was allowed to crystallize for 0.5 hour, collected by filtration
and recrystallized from i-PrOH.
Procedure 12
Synthesis of N.sup..alpha. -(.omega.-(benzylthio)alkyl)amines
(Bzl-S--(CH.sub.2).sub.n --NH.sub.2) (known compounds).
Hydrazinolysis of the phthalimide group was performed by refluxing 0.09
mole of N.sup..alpha. -(.omega.-(benzylthio)alkylene)-phthalimide
(Procedure 11) with 120 mL 1M solution of hydrazine hydrate in ethanol
(diluted with additional 220 mL ethanol) for 2 hours. The formed
precipitate was collected by filtration and hydrolyzed with 180 mL 2N HCl
at 50.degree. C. for 0.5 hour. The water was evaporated in vacuo and the
crude hydrochloride dissolved in 50 mL 25% ammonia solution. The free
amine was extracted with DCM (4.times.100 mL) and the organic phase was
washed with brine (2.times.100 mL), dried over MgSO.sub.4 and the solvent
evaporated in vacuo. The crude N-(.omega.-(benzyl-thio)alkyl)amine was
distilled under reduced pressure, and appeared as colorless oil, which
could be kept refrigerated under nitrogen for prolonged periods.
Procedure 13
Synthesis of (R) and (S) N.sup..alpha. -(.omega.-(benzylthio)alkylene)amino
acids methyl esters ((R) or (S) (Bzl-S--(CH.sub.2).sub.n
--NH--CH(R)--COOMe)).
To a solution of 15 mmol N-(.omega.-(benzylthio)alkyl)amine and 15 mmol
DIEA in 55 mL DCM was added dropwise a solution of 15 mmol (R) or (S)
.alpha.-hydroxy acid methyl ester triflate (Procedure 5) in 55 mL DCM at
0.degree. C. The reaction mixture was then stirred at room temperature for
18 h. The mixture was then diluted with 100 mL DCM and washed with water
(3.times.100 mL). The crude product was cleaned on a silica-gel column
with DCM:MeOH(99:1), and was further crystallized from DIE:hexane.
In the case of Glycine, bromoacetic acid esters were suitable starting
materials. Identical results were obtained when both these substrates were
reacted with N-(.omega.-(benzylthio) alkyl)amines.
Procedure 14
Synthesis of (R) or (S) Boc-N.sup..alpha.
-(.omega.-(benzylthio)alkylene)amino acids ((R) or (S)
(Bzl-S--(CH.sub.2).sub.n --N(Boc)--CH(R)--COOH)).
10 mmol (R) or (S) N-(.omega.-(benzylthio)alkylene) amino acid methyl ester
was dissolved in 50 mL 1,4-dioxane and 50 mL 1N NaOH were added. The
mixture was stirred at room temperature overnight. The disappearance of
the starting material was followed by TLC (Silica gel+F.sub.254,
CHCl.sub.3 :MeOH-1:4). When all the ester was hydrolyzed, 50 mL water were
added, followed by 30 mmol Boc.sub.2 O. The mixture was stirred overnight,
then the dioxane was evaporated in vacuo, the mixture was cooled in an
ice-water bath, covered with 100 mL EtAc and acidified with saturated
KHSO.sub.4 to pH 2-3. The layers were separated, and the aqueous layer was
extracted with additional 2.times.100 mL EtOAc. The organic layer was
washed with water (2.times.100 mL), dried over MgSO.sub.4 and the solvent
evaporated in vacuo. The crude product was cleaned on a silica-gel column
with DCM:MeOH-99:1 or crystallized from DIE:hexane.
Procedure 15
Synthesis of dipeptides (S,S)-Boc-amino acid-N.sup..alpha.
-(.omega.-(benzylthio)alkylene)amino acids esters.
A solution of 1.1 mmol Boc-amino acid, 1 mmol N.sup..alpha.
-(.omega.-(benzylthio)alkylene) amino acid ester, 1.1 mmol BOP and 3 mmol
DIEA in 10 mL DCM was stirred at room temperature for 2 hours. Then the
mixture was diluted with 40 mL of DCM and washed successively with
saturated KHSO.sub.4 (3.times.100 mL), saturated KHCO.sub.3 (3.times.100
mL) and brine (2.times.100 mL), dried over MgSO.sub.4 and the solvent
evaporated in vacuo. The crude product was cleaned on a silica-gel column
with DCM:MeOH-99:1 or crystallized from DIE:hexane.
Procedure 16
Synthesis of N.sup..alpha. -Bzl .omega.-amino acids t-butyl esters
(Bzl--NH--(CH.sub.2).sub.n --COO-t-Bu).
A solution of 0.05 mole of amino acid t-butyl ester acetate in 200 mL
H.sub.2 O was acidified to pH 2 with AcOH, washed with PE (70 mL X5)
cooled and the pH adjusted to 9 by NH.sub.4 OH 25%. The free amino acid
t-butyl ester was extracted with i-Pr:CHCl (3:1, 3.times.100 mL). The
combined extracts were dried on Na.sub.2 SO.sub.4 and evaporated to
dryness under vacuum. The benzylation reaction was performed according to
Procedure 2.
Procedure 17
Synthesis of N.sup..alpha. -(Bzl)(.omega.t-Bu carboxy alkylene)glycine Bzl
ester (N-(Bzl) (CH.sub.2).sub.n --COO-t-Bu)CH.sub.2 --COO-Bzl)
To a stirred solution of 0.015 mole of N-Bzl .omega.-amino acid t-butyl
ester in 10 mL DMF at 0.degree. C. were added 2.61 mL of DIEA and 2.38 mL
of benzyl bromo acetate. The reaction mixture was stirred 30 min at
0.degree. C. and 3 h at room temperature. After the addition of 200 mL of
ether, the precipitate was removed by filtration and the organic phase
washed with H.sub.2 O (3.times.80 mL), 1N HCl (3.times.80 mL), H.sub.2 O
(3.times.80 mL), dryed over Na.sub.2 SO.sub.4 and evaporated to dryness
under vacuum. The resulting oil was dried under vacuum.
METHODS
ANALYTICAL TLC was performed on TLC plates of silica gel E (Merck
F.sub.254), using the following solvent systems:
METHOD A DCM:MeOH:AcOH 16:4:0.5
METHOD B PE:EtOAc 1:1
METHOD C PE:EtOAc 4:1
METHOD D CHCl.sub.3 :EtOAc 4:1
METHOD E PE:EtOAc 9:1
METHOD F CHCl.sub.3 :EtOAc 19:1
METHOD G ANALYTICAL REVERSE PHASE HPLC
Column Merck LICHROCART RP-18 5 .mu.m, 250 .times. 4 mm.
Mobile Phases A = 0.1% TFA in H.sub.2 O
B = 0.1% TFA in ACN
Gradient T = 0-5 min A(75%), B(25%)
T = 30 min A(50%), B(50%)
T = 40 min A(100%), B(0%)
T = 50 min A(100%), B(0%)
Flow = 1 mL/minute Temperature = 23.degree. C.
METHOD H--SEMIPREPARATIVE REVERSE PHASE HPLC
The crude peptide was dissolved in MeOH (1 mL) and chromatographed using
reverse phase semipreparative HPLC with the following conditions:
Column Merck Hibar LICHROSORB RP-18 7 .mu.m, 250 .times.
410 mm
Mobile Phases A = 0.1% TFA in H.sub.2 O
B = 0.1% TFA in ACN
Gradient T = 0-10 min A(80%), B(20%)
T = 60 min A(100%), B(0%)
T = 70 min A(100%), B(0%)
Flow = 4 mL/minute Temperature = 23.degree. C.
METHOD J NINHYDRIN TEST (NIN. TEST)
The test was performed according to Kaiser et al. Anal. Biochem., 34:595
(1970) which is incorporated herein by reference in its entirety. Test was
considered negative when the resin did not change color after heating to
11.degree. C. for 2 minutes with the test mixture. Test was considered
positive or slightly positive when the resin was dark or faint purple
after heating to 110.degree. C. for 2 minutes with the test mixture.
METHOD K--QUANTITATIVE PICRIC ACID TEST
Picric acid test was performed after removal of the Fmoc protecting group
from the amino acid preceding the coupling of protected
N.alpha.(.omega.-alkylene) building unit. This absorbance was taken as
100% free amines. After coupling the test was used to check yield of %
coupling by comparison.
The resin 0.1 mmole was treated according to steps 1-8, Table 1. After step
8 the resin was introduced into a centrifuge tube and shaken with 40 mL of
5% DIEA:95% DCM for ten minutes. The resin was centrifuged 5 minutes and 4
mL of the solution were pipette into 40 mL. EtOH and the absorbance at 358
nm measured. This procedure was repeated 3 times and the average
absorbance calculated.
TABLE 6
PROCEDURE FOR 0.1 mMOLE SCALE
STEP SOLVENT/ VOLUME TIME REPEAT
NO. REAGENT (ML) (MIN) (XS) COMMENT
1 NMP 10 1 3
2 DCM 10 1 2
3 Picric acid 10 1 2
0.05M/DCM
4 DMF 10 0.5 10
5 NMP 10 1 2
6 NMP 10 2 3
7 10% EtOH/DCM 10 1 1
8 DCM 10 1 2
COMPOUND 1
N-Boc diamino ethane (known compound)
A solution of 33.4 mL of ethylene diamine in CHCl.sub.3 and 10.91 g of
Boc.sub.2 O were used (Procedure 1). Yield 97% of colorless oil.
TLC (Method A) Rf 0.2-0.24 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 2
N-Boc 1,3 diamino propane (known compound).
A solution of 41.7 mL of 1,3 diamino propane in CHCl.sub.3 and 10.91 g of
Boc.sub.2 O were used (Procedure 1). Yield 96% of colorless oil.
TLC (Method A) Rf 0.27-0.3 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 3
N-Boc 1,4 diamino butane (known compound).
A solution of 44.08 g of 1,4 diamino butane and 10.91 g of Boc.sub.2 O were
used (Procedure 1). Yield 98% of white oil.
TLC (Method A) Rf 0.32-0.35 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 4
N-Boc 1,6 diamino hexane (known compound).
A solution of 58.10 g of 1,6 diamino hexane in CHCl.sub.3 and 10.91 g of
Boc.sub.2 O were used (Procedure 1). Yield 70% of colorless oil after
purification on a silica gel column and elution with CHCl.sub.3 --MeOH
(4:1).
TLC (Method A) Rf 0.50-0.54 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 5
N-Boc, N-Bzl 1,2 diamino ethane
A solution of 8.01 g of Boc ethylene diamine (COMPOUND 1) was used
(Procedure 2). Yield 65% of colorless oil.
TLC (Method A) Rf 0.62-0.65 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 6
N-Boc, N-Bzl, 1,3 diamino propane.
A solution of 8.71 g of N-Boc 1,3 diamino propane (COMPOUND 2) was used
(Procedure 1). Yield 75% of colorless.
TLC (Method A) Rf 0.63-0.68 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 7
N-Boc, N-Bzl 1,4 diamino butane
A solution of 9.41 g of N-Boc 1,4 diamino butane (Compound 3) was used
(Procedure 1). Yield 63% of white oil.
TLC (Method A) Rf 0.65-0.72 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
COMPOUND 8
N-Boc, N-Bzl 1,6 diamino hexane
A solution of 10.82 g of N-Boc 1,6 diamino hexane (Compound 4) was used
(Procedure 1). The ethyl acetate solution after extraction was dried over
Na.sub.2 SO.sub.4 and evaporated under vacuum to dryness. The remaining
crude product was dissolved in 400 mL chloroform and washed with 0.5 N HCl
(3.times.80 mL, 0.12 mole), water (2.times.100 mL) dried over Na.sub.2
SO.sub.4 and evaporated to dryness. Then 200 mL of ether was added. The
precipitate was filtered, washed with ether (3.times.50 mL) and dried
under vacuum.
Yield 70% of white solid mp 150-152.degree. C.; TLC (Method A) Rf 0.8 (one
spot).
To remove HCl from product with n=6, the HCl salt was dissolved in
CHCl.sub.3, washed with an alkali solution (0.5% NH.sub.4 OH), dried over
Na.sub..ltoreq. SO.sub.4 and evaporated to dryness. NMR (CDCl.sub.3) in
agreement with the title compound.
COMPOUND 9
(S)-3-Phenylacetic acid methyl ester (known compound)
A suspension of 10.8 g of (S)-3-Phenylacetic acid in 100 mL ether was
treated with diazomethane (Procedure 4).
Yield 85%. TLC (Method B) Rf 0.6-0.65 (one spot); (.alpha.).sub.D =+3,3
(c=1, MeOH); NMR (CDCl.sub.3) in agreement with the title compound.
COMPOUND 10
(R)-3-Phenylacetic acid methyl ester (known compound).
A suspension of 10.8 g (R)-3-Phenylacetic acid in 100 mL ether was treated
with diazomethane (Procedure 4). Yield 84%.
TLC (Method B) Rf 0.6-0.65 (one spot); (.alpha.).sub.D =-3,3 (c=1, MeOH);
NMR (CDCl.sub.3) in agreement with the title compound.
COMPOUND 11
(S)-O-Trf-3-Phenylacetic acid methyl ester
To a cooled solution of Trf.sub.2 O and pyridine in dry DCM (Procedure 5),
a solution of 5.4 g of (S)-3-Phenylacetic acid methyl ester was added.
After the workup (Procedure 5), the yield was 74%. The product was used
immediately or kept in a cold desiccator under Ar.
COMPOUND 12
(R)-O-Trf-3-Phenylacetic acid methyl ester
To a cooled solution of Trf.sub.2 O and pyridine in dry DCM (Procedure 5),
a solution of 5.4 g of (R)-3-Phenylacetic acid methyl ester was added.
After the workup (Procedure 5), the yield was 74%. The product was used
immediately or kept in a cold desiccator under Ar.
COMPOUND 13
N.sup..alpha. (Bzl)(2-Boc-amino ethylene)(R)Phenylalanine methyl ester
A solution of 6.24 g of (S)-O-Trf-3-Phenyllactic acid methyl ester in dry
DCM (Compound 11) was added to a solution of 5.51 g of N-Boc,
N-Bzl-diamino ethane (Compound 5) in dry DCM (Procedure 6). Yield 69.2%
(.alpha.).sub.D =+64.0 (c=1, MeOH); TLC (Method C) Rf=0.41 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 14
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(S)Phenylalanine methyl ester
A solution of 6.24 g of (R)-O-Trf-3-Phenyllactic acid methyl ester in dry
DCM (Compound 12) was added to a solution of 5.82 g of N-Boc,
N-Bzl-diamino propane (Compound 6) in dry DCM (Procedure 6). Yield 67.7%
(.alpha.).sub.D =-55.8 (c=1, MeOH); TLC (Method C) Rf=0.38 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 15
N.sup..alpha. (Bzl)(4-Boc-amino butylene)(S)Phenylalanine methyl ester
A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methyl ester in dry
DCM (Compound 12) was add to a solution of 6.12 g of N-Boc, N-Bzl-diamino
butane (Compound 7) in dry DCM (Procedure 6). Yield 58.6%
(.alpha.).sub.D =-62.6 (c=1, MeOH); Rf (Method C) 0.42 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 16
N.sup..alpha. (Bzl)(6-Boc-amino hexylene)(S)Phenylalanine methyl ester
A solution of 6.24 g of O-Trf-(R)-3-Phenyllactic acid methyl ester in dry
DCM (Compound 12) was add to a solution of 6.74 g of N-Boc, N-Bzl-diamino
hexane (Compound 8) in dry DCM (Procedure 6). Yield 78.9%
(.alpha.).sub.D =-60.0 (c=1, MeOH); TLC (Method C) Rf=0.47 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 17
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(R)Phenylalanine methyl ester
A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methyl ester in dry
DCM (Compound 11) was add to a solution of 5.82 g of N-Boc, N-Bzl-diamino
propane (Compound 6) in dry DCM (Procedure 6). Yield 51.5%
(.alpha.).sub.D =+58.8 (c=1, MeOH); TLC (Method C) Rf=0.35 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 18
N.sup..alpha. (Bzl)(4-Boc-amino butylene)(R)Phenylalanine methyl ester
A solution of 6.24 g of O-Trf-(S)-3-Phenyllactic acid methyl ester in dry
DCM (Compound 11) was add to a solution of 6.12 g of N-Boc, N-Bzl-diamino
butane (Compound 7) in dry DCM (Procedure 6). Yield 66.8%
(.alpha.).sub.D =+59.0 (c=1, MeOH); TLC (Method C) Rf=0.33 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 19
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(S)Phenylalanine
A solution of 6.61 g of N.sup..alpha. (Bzl)(3-Boc-amino propylene)
(S)Phenylalanine methyl ester in MeOH (Compound 14) was hydrolyzed by NaOH
7.5N (Procedure 7). Yield 89.5%
(.alpha.).sub.D =-24.0 (c=1, MeOH); TLC (Method B) Rf=0.16 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 20
N.sup..alpha. (Bzl)(4-Boc-amino butylene)(S)Phenylalanine
A solution of 6.61 g of N.sup..alpha. (Bzl)(4-Boc-amino butylene)
(S)Phenylalanine methyl ester in MeOH (Compound 15) was hydrolyzed by NaOH
7.5N (Procedure 7). Yield 73.5%
(.alpha.).sub.D =-12.0 (c=1, MeOH); TLC (Method D) Rf=0.6 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 21
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(R)Phenylalanine
A solution of 6.40 g of N.sup..alpha. (Bzl)(3-Boc-amino propylene)
(R)Phenylalanine methyl ester in MeOH (Compound 17) was hydrolyzed by NaOH
7.5N (Procedure 7). Yield 100%
(.alpha.).sub.D =+15.33 (c=1, MeOH); TLC (Method B) Rf=0.38 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 22
N.sup..alpha. (Bzl)(4-Boc-amino butylene)(R)Phenylalanine
A solution of 6.61 g of N.sup..alpha. (Bzl)(4-Boc-amino butylene)
(R)Phenylalanine methyl ester in MeOH (Compound 18) was hydrolyzed by NaOH
7.5N (Procedure 7). Yield 100%
(.alpha.).sub.D =+12.0 (c=1, MeOH); TLC (Method D) Rf=0.54 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 23
N.sup..alpha. (3-Boc-amino propylene)(S)Phenylalanine HCl
A solution of 5.39 g of N.sup..alpha. (Bzl)(3-Boc-amino propylene)
(S)Phenylalanine (Compound 19) in MeOH-DMF was hydrogenated on Pd/C
(Procedure 8). Yield 86.1%
TLC (Method A) Rf=0.51 (one spot); NMR (D.sub.2 O+Na.sub.2 CO.sub.3) in
agreement with the title compound.
COMPOUND 24
N.sup..alpha. (4-Boc-amino butylene)(S)Phenylalanine HCl
A solution of 5.56 g of N.sup..alpha. (Bzl)(4-Boc-amino butylene)
(S)Phenylalanine (Compound 20) in MeOH-DMF was hydrogenated on Pd/C
(Procedure 8). Yield 79.25%
TLC (Method A) Rf=0.50 (one spot); NMR (D.sub.2 O+Na.sub.2 CO.sub.3) in
agreement with the title compound.
COMPOUND 25
N.sup..alpha. (3-Boc-amino propylene)(R)Phenylalanine HCl
A solution of 5.39 g of N.sup..alpha. (Bzl)(3-Boc-amino propylene)
(R)Phenylalanine (Compound 21) in MeOH-DMF was hydrogenated on Pd/C
(Procedure 8). Yield 75.5%
TLC (Method A) Rf=0.51 (one spot); NMR (D.sub.2 O+Na.sub.2 CO.sub.3) in
agreement with the title compound.
COMPOUND 26
N.sup..alpha. (4-Boc-amino butylene)(R)Phenylalanine HCl
A solution of 5.56 g of N.sup..alpha. (Bzl)(4-Boc-amino butylene)
(R)Phenylalanine (Compound 22) in MeOH-DMF was hydrogenated on Pd/C
(Procedure 8). Yield 73.85%
TLC (Method A) Rf=0.50 (one spot); NMR (D.sub.2 O+Na.sub.2 CO.sub.3) in
agreement with the title compound.
EXAMPLE 5
COMPOUND 27
N.sup..alpha. (Fmoc)(3-Boc-amino propylene)(S)Phenylalanine
A solution of 2.51 g of N.sup..alpha. (3-Boc-amino propylene)
(S)Phenylalanine.HCl (Compound 23) in H.sub.2 O-ACN was reacted with
FmocOSu (Procedure 9). Yield 64.84%
TLC (Method D) Rf=0.74 (one spot); (.alpha.).sub.D =-87 (c=1, MeOH); HPLC
(Method G) 92%; NMR (CDCl.sub.3) in agreement with the title compound.
EXAMPLE 6
COMPOUND 28
N.sup..alpha. (Fmoc)(3-Boc-amino propylene)(R)Phenylalanine
A solution of 2.51 g of N.sup..alpha. (3-Boc-amino propylene)
(R)Phenylalanine.HCl (Compound 25) in H.sub.2 O-ACN was reacted with
FmocOSu (Procedure 9). Yield 61.56%
TLC (Method D) Rf=0.62 (one spot); (.alpha.).sub.D =+79.6 (c=1, MeOH); HPLC
(Method G) 94%; NMR (CDCl.sub.3) in agreement with the title compound.
EXAMPLE 7
COMPOUND 29
N.sup..alpha. (Fmoc)(4-Boc-amino butylene)(S)Phenylalanine
A solution of 2.61 g of N.sup..alpha. (4-Boc-amino butylene)
(S)Phenylalanine.HCl (Compound 24) in H.sub.2 O-ACN was reacted with
FmocOSu (Procedure 9). Yield 56%
TLC (Method D) Rf=0.64 (one spot); HPLC (Method G) 89%; NMR (CDCl.sub.3) in
agreement with the title compound.
COMPOUND 30
O-Trf-(S)-Lactic acid methyl ester
To a cooled solution of Trf.sub.2 O and pyridine in DCM (Procedure 5), a
solution of 2.9 mL of (S)lactic acid methyl ester was added. After the
workup (Procedure 5), the yield was 70%. The product was used immediately
or kept in a cold desiccator under Ar.
COMPOUND 31
O-Trf-(R)-Lactic acid methyl ester
To a cooled solution of Trf.sub.2 O and pyridine in DCM (Procedure 5), a
solution of 2.9 mL of (R) lactic acid methyl ester was added. After the
workup (Procedure 5), the yield was 70%. The product was used immediately
or kept in a cold desiccator under Ar.
COMPOUND 32
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(S)Alanine methyl ester
A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dry DCM
(Compound 31) was add to a solution of 5.82 g of N-Boc, N-Bzl-diamino
propane (Compound 6) in dry DCM (Procedure 6). Yield 69.5%
(.alpha.).sub.D =-6.6 (C=1, MeOH); TLC (Method C) Rf=0.42 (one spot); TLC
(Method D) Rf=0.92 (one spot); TLC (Method E) Rf=0.13 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 33
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(R)Alanine methyl ester
A solution of 4.72 g of O-Trf-(S-lactic acid methyl ester in dry DCM
(Compound 30) was add to a solution of 5.82 g of N-Boc, N-Bzl-diamino
propane (Compound 6) in dry DCM (Procedure 6). Yield 71%
(.alpha.).sub.D =+6.53 (C=1, MeOH); TLC (Method C) Rf=0.42 (one spot); TLC
(Method D) Rf=0.93 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 34
N.sup..alpha. (Bzl)(6-Boc-amino hexylene)(S)Alanine methyl ester
A solution of 4.72 g of O-Trf-(R)-lactic acid methyl ester in dry DCM
(Compound 31) was add to a solution of 6.74 g of N-Boc, N-Bzl-diamino
hexane (Compound 8) in dry DCM (Procedure 6). Yield 81.42%
(.alpha.).sub.D =-6.76 (C=1, MeOH); TLC (Method D) Rf=0.95 (one spot); TLC
(Method E) Rf=0.26 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 35
N.sup..alpha. (Bzl)(2-Boc-amino propylene)(S)Alanine
A solution of 5.25 g of N.sup..alpha. (Bzl)(2-Boc-amino ethylene)
(S)Alanine methyl ester in MeOH (Compound 32) was hydrolyzed by NaOH 7.5N
(Procedure 7). Yield 100% of white solid, mp 64.degree. C.
(.alpha.).sub.D =+0.5 (C=1, MeOH); TLC (Method A) Rf=0.64 (one spot); TLC
(Method D) Rf=0.47 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 36
N.sup..alpha. (Bzl)(6-Boc-amino hexylene)(S)Alanine
A solution of 5.88 g of N.sup..alpha. (Bzl)(6-Boc-amino hexylene)
(S)Alanine methyl ester (Compound 34) in MeOH was hydrolyzed by NaOH 7.5N
(Procedure 7). Yield 100%
(.alpha.).sub.D =+0.7 (C=1, MeOH); TLC (Method D) Rf=0.51 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 37
N.sup..alpha. (Bzl)(3-Boc-amino propylene)(R)Alanine
A solution of 5.25 g of N.sup..alpha. (Bzl)(2-Boc-amino ethylene)
(R)Alanine methyl ester (Compound 35) in MeOH was hydrolyzed by NaOH 7.5N
(Procedure 7). Yield 100%
(.alpha.).sub.D =-0.5 (C=1, MeOH); TLC (Method D) Rf=0.51 (one spot); NMR
(CDCl.sub.3) in agreement with the title compound.
COMPOUND 38
N.sup..alpha. (3-Boc-amino propylene)(S)Alanine.HCl
A solution of 4.47 g of N.sup..alpha. (Bzl)(3-Boc-amino propylene)
(S)Alanine (Compound 35) in MeOH was hydrogenated on Pd/C (Procedure 8).
Yield 75%
TLC (Method A) Rf=0.42 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 39
N.sup..alpha. (6-Boc-amino hexylene)(S)Alanine. HCl
A solution of 5 g of N.sup..alpha. (Bzl)(4-Boc-amino hexylene) (S)Alanine
(Compound 36) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield
64.5% of white solid, mp 134-136.degree. C.
TLC (Method A) Rf=0.39 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 40
N.sup..alpha. (3-Boc-amino propylene)(R)Alanine. HCl
A solution of 5 g of N.sup..alpha. (Bzl)(4-Boc-amino propylene) (R)Alanine
(Compound 37) in MeOH-DMF was hydrogenated on Pd/C (Procedure 8). Yield
79.1%.
TLC (Method A) Rf=0.39 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
EXAMPLE 8
COMPOUND 41
N.sup..alpha. (Fmoc)(3-Boc-amino propylene)(S)Alanine
A solution of 2.82 g of N.sup..alpha. (Bzl)(4-Boc-amino propylene)
(S)Alanine .HCl (Compound 38) in H.sub.2 O-ACN was reacted with FmocOSu
(Procedure 9). Yield 75% of white solid, mp 70-72.degree. C.
TLC (Method D) Rf=0.65 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
Elemental Analysis: % C % H % N
Found: 66.40 6.78 5.63
Calc: 66.65 6.88 5.93
EXAMPLE 9
COMPOUND 42
N.sup..alpha. (Fmoc)(6-Boc-amino hexylene)(S)Alanine
A solution of 3.25 g of N.sup..alpha. (Bzl)(4-Boc-amino hexylene)
(S)Alanine .HCl (Compound 39) in H.sub.2 O-ACN was reacted with FmocOSu
(Procedure 9). Yield 72.8% of white solid, mp 70-72.degree. C.
TLC (Method D) Rf=0.7 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
Elemental Analysis: % C % H % N
Found: 68.37 7.40 5.23
Calc: 68.21 7.50 5.49
HPLC (Method G) 90%
EXAMPLE 10
COMPOUND 43
N.sup..alpha. (Fmoc)(3-Boc-amino propylene)(R)Alanine
A solution of 2.82 g of N.sup..alpha. (Bzl)(4-Boc-amino propylene)
(S)Alanine .HCl (Compound 40) in H.sub.2 O-ACN was reacted with FmocOSu
(Procedure 9). Yield 75.9% of white solid, mp 70-72.degree. C.
TLC (Method D) Rf=0.5 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
Elemental Analysis: % C % H % N
Found: 66.4 6.78 5.63
Calc: 66.65 6.88 5.93
EXAMPLE 11
COMPOUND 44
N-(2-(benzylthio)ethylene)glycine ethyl ester
The title compound was prepared according to procedure 13 from ethyl bromo
acetate.
Yield 75% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. Elemental analysis-calculated: C-61.16, H-7.70, N-3.96; found:
C-61.45, H-8.03, N-3.49.
COMPOUND 45
N-(3-(benzylthio)propylene)glycine methyl ester
The title compound was prepared according to procedure 13 from methyl bromo
acetate.
Yield 74% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound.
EXAMPLE 12
COMPOUND 46
N-(2-(benzylthio)ethylene)(S)leucine methyl ester
The title compound was prepared according to procedure 13 from the Triflate
of (R) leucine methyl ester (Procedure 5).
Yield 70% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. Elemental analysis-calculated: C-65.05, H-8.53, N-4.74; found:
C-66.29, H-9.03, N-4.49. (a).sub.D14 =-51.2.degree. C. (C 0.94,DCM).
EXAMPLE 13
COMPOUND 47
N-(3-(benzylthio)propylene)(S)leucine methyl ester
The title compound was prepared according to procedure 13 from the Triflate
of (R)leucine methyl ester (Procedure 5).
Yield 60% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. Elemental analysis-calculated: C-65.98, H-8.79, N-4.53; found:
C-67.09, H-9.20, N-4.54. (a).sub.D23 =-17.4.degree. (C 1.44, DCM).
COMPOUND 48
N-(2-(benzylthio)ethylene)(S)phenylalanine methyl ester
The title compound was prepared according to procedure 13 from the Triflate
of (R)phenyl lactic acid methyl ester (Procedure 5).
Yield 82% of white crystals. m.p.=48-49.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. Elemental analysis-calculated: C-69.27,
H-7.04, N-4.25; found: C-69.55, H-7.21, N-4.08. (a).sub.D14 =-23.3.degree.
(C=1.01, DCM).
COMPOUND 49
N-(3-(benzylthio)propylene)(S)phenylalanine methyl ester
The title compound was prepared according to procedure 13 from the Triflate
of (R)phenyl lactic acid methyl ester (Procedure 5).
Yield 71% of white crystals. m.p.=38-39.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. Elemental analysis-calculated: C-69.94,
H-7.34, N-4.08; found: C-69.66, H-7.39, N-4.37. (a).sub.D26 =+2.0.degree.
(C 1.00, DCM).
COMPOUND 50
N-(4-(benzylthio)butylene)(S)phenylalanine methyl ester
The title compound was prepared according to procedure 13 from the Triflate
of (S)phenyl lactic acid methyl ester (Procedure 5).
Yield 81% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. Elemental analysis-calculated: C-70.55, H-7.61, N-3.92; found:
C-70.51, H-7.69, N-4.22. (a).sub.D26 =+4.9.degree. (C 1.00, DCM).
EXAMPLE 14
COMPOUND 51
Boc-N-(2-(benzylthio)ethylene)glycine
The title compound was prepared from Compound 44 by hydrolysis according to
Procedure 11.
Yield 88% of white crystals. m.p.=71-72.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. Elemental analysis-calculated: C-59.05,
H-7.12, N-4.30; found: C-59.39, H-7.26, N-4.18.
EXAMPLE 15
COMPOUND 52
Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine
The title compound was prepared from Compound 48 by hydrolysis according to
Procedure 11.
Yield 78% of white crystals. m.p.=82-83.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. (a).sub.D25 =-105.9.degree. (C 1.01,
DCM).
EXAMPLE 16
COMPOUND 53
Boc-N-(3-(benzylthio)propylene)(S)phenylalanine
The title compound was prepared from Compound 49 by hydrolysis according to
Procedure 11.
Yield 99% of white crystals. m.p.=63-64.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. (a).sub.D25 =-87.4.degree. (C 1.01,
DCM).
EXAMPLE 17
COMPOUND 54
Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester
Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethyl ester
(Compound 44) according to Procedure 12.
Yield 32% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. Elemental analysis-calculated: C-64.77, H-7.25, N-5.60; found:
C-64.39, H-7.02, N-5.53. (a).sub.D16 =+4.5.degree. (C 0.88, DCM).
EXAMPLE 18
COMPOUND 55
Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalanine methyl ester
Boc-L-Phe was coupled to N-(2-(benzylthio)ethylene) (S)phenylalanine methyl
ester (Compound 48) according to Procedure 12.
Yield 46% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. (a).sub.D26 =-115.9.degree. (C 1.0, CHCl.sub.3).
COMPOUND 56
N-Bzl-.beta.-alanine t-butyl ester
A solution of 6.16 g of .beta.-alanine t-butyl ester acetate in 150 mL
water was reacted with benzaldhyde (Procedure 2) to give 4.5 g, 64.5%
yield
TLC (Method A) Rf=0.78 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 57
N-Bzl-.gamma.-amino butyric acid t-butyl ester
A solution of 6.58 g of .gamma.-aminobutyric acid t-butyl ester acetate in
150 mL water was reacted with benzaldhyde (Procedure 2) to give 4.24 g,
57.9% yield
TLC (Method A) Rf=0.74 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 58
N.sup..alpha. (Bzl)(2-t-butyl carboxy ethylene)glycine benzyl ester
A solution of 3.53 g of N-Bzl-.beta.-alanine t-butyl ester (Compound 56) in
DMF was reacted with 2.61 mL benzyl bromoacetate (Procedure 17). Yield
86.9%
TLC (Method F) Rf=0.95 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 59
N.sup..alpha. (Bzl)(3-t-butyl carboxy propylene)glycine benzyl ester
A solution of 3.53 g of N-Bzl-.gamma.-aminobutyric acid t-butyl ester
(Compound 57) in DMF was reacted with 2.61 mL benzyl bromoacetate
(Procedure 17). Yield 83%
TLC (Method F) Rf=0.92 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 60
N.sup..alpha. (2-t-butyl carboxy ethylene)glycine
A solution of N.sup..alpha. (Bzl)(2-t-butyl carboxy ethylene)glycine benzyl
ester (Compound 58) in MeOH was hydrogenated (Procedure 8). Yield 87.8%
TLC (Method A) Rf=0.56 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 61
N.sup..alpha. (3-t-butyl carboxy propylene)glycine
A solution of N.sup..alpha. (Bzl)(3-t-butyl carboxy propylene)glycine
benzyl ester (Compound 59) in MeOH was hydrogenated (Procedure 8). Yield
94%
TLC (Method A) Rf=0.3 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
EXAMPLE 19
COMPOUND 62
N.sup..alpha. (Fmoc)(2-t-butyl carboxy ethylene)glycine
A solution of N.sup..alpha. (2-t-butyl carboxy ethylene)glycine (Compound
60) in H.sub.2 O:Et.sub.3 N was reacted with FmocOSu (Procedure 9). Yield
90%
TLC (Method D) Rf=0.5 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
Elemental Analysis: % C % H % N
Found: 67.38 6.34 3.11
Calc: 67.75 6.40 3.29
EXAMPLE 20
COMPOUND 63
N.sup..alpha. (Fmoc)(3-t-butyl carboxy propylene)glycine
A solution of N.sup..alpha. (3-t-butyl carboxy propylene)glycine (Compound
61) in H.sub.2 O:Et.sub.3 N was reacted with FmocOSu (Procedure 9). Yield
82%
TLC (Method D) Rf=0.58 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
Elemental Analysis: % C % H % N
Found: 68.29 6.83 3.88
Calc: 68.32 6.65 3.19
COMPOUND 64
(R)-O-Trf-3-Phenyllactic acid benzyl ester
To a cooled solution of Trf.sub.2 O and pyridine in dry DCM (Procedure 5),
a solution of 5.3 g of (R)-3-Phenyllactic acid benzyl ester was added.
After the workup (Procedure 5), the yield was 91.43%. The product was used
immediately or kept in a cold desiccator under Ar.
COMPOUND 65
N.sup..alpha. (Bzl)(2-t-butyl carboxy ethylene)(S)Phenylalanine benzyl
ester
A solution of 5.48 g of N-Bzl-.beta.-alanine t-butyl ester (Compound 56) in
DCM was reacted with 7.35 g of (R)-O-Trf-3-Phenyllactic acid benzyl ester
(COMPOUND 64) in dry DCM (Procedure 6). After workup the crude product was
purified by flash chromatography. PE:EtOAc (4:1) 1.5 L. After solvent
evaporation under vacuum, the product was dried under vacuum.
Yield 71.5%; TLC (Method C) Rf=0.77 (one spot); (.alpha.).sub.D =-62.7
(C=1, MeOH); NMR (CDCl.sub.3) in agreement with the title compound.
COMPOUND 66
N.sup..alpha. (2-t-butyl carboxy ethylene)(S)Phenylalanine
A solution of 6.3 g of N.sup..alpha. (Bzl)(2-t-butyl carboxy
ethylene)(S)Phenylalanine benzyl ester (Compound 65) in MeOH was
hydrogenated (Procedure 8). Yield 48.6%
TLC (Method A) Rf=0.52-0.54 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
EXAMPLE 21
COMPOUND 67
N.sup..alpha. (Fmoc)(2-t-butyl carboxy ethylene)(S)Phenylalanine
A solution of 2.13 g of N.sup..alpha. (2-t-butyl carboxy
ethylene)(S)Phenylalanine (Compound 66) in H.sub.2 O:Et.sub.3 N was
reacted with FmocOSu (Procedure 9). Yield 38%
TLC (Method D) Rf=0.77 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
Elemental Analysis: % C % H % N
Found: 71.92 639 2.87
Calc: 72.21 6.45 2.72
HPLC (Method G) 93%
COMPOUND 68
N.sup..alpha. (Bzl)(2-Boc amino ethylene)glycine benzyl ester
Elemental analysis-calculated: C-59.05, H-7.12, N-4.30; found: C-59.39,
H-7.26, N-4.18.
EXAMPLE 15
COMPOUND 52
Boc-N-(2-(benzylthio)ethylene)(S)phenylalanine
The title compound was prepared from Compound 48 by hydrolysis according to
Procedure 11.
Yield 78% of white crystals. m.p.=82-83.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. (a).sub.D25 =-105.9.degree. (C 1.01,
DCM).
EXAMPLE 16
COMPOUND 53
Boc-N-(3-(benzylthio)propylene)(S)phenylalanine
The title compound was prepared from Compound 49 by hydrolysis according to
Procedure 11.
Yield 99% of white crystals. m.p.=63-64.degree. C. NMR (CDCl.sub.3) in
agreement with the title compound. (a).sub.D25 =-87.4.degree. (C 1.01,
DCM).
EXAMPLE 17
COMPOUND 54
Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)glycine ethyl ester
Boc-L-Phe was coupled to N-(2-(benzylthio)-ethylene)glycine ethyl ester
(Compound 44) according to Procedure 12.
Yield 32% of colorless oil. NMR (CDCl.sub.3) in agreement with the title
compound. Elemental analysis-calculated: C-64.77, H-7.25, N-5.60; found:
C-64.39, H-7.02, N-5.53. (a).sub.D16 =+4.5.degree. (C 0.88, DCM).
EXAMPLE 18
COMPOUND 55
Boc-L-phenylalanyl-N-(2-(benzylthio)-ethylene)(S)phenylalanine methyl ester
NMR (CDCl.sub.3) in agreement with the title compound.
COMPOUND 73
N.sup..alpha. (3-Boc amino propylene)glycine
A solution of 0.025 mole of N.sup..alpha. (Bzl)(3-Boc amino
propylene)glycine benzyl ester (Compound 69) in 60 mL MeOH was
hydrogenated (Procedure 8). Yield 74% of white solid. mp 214-6.degree. C.
TLC (Method A) Rf=0.27 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 74
N.sup..alpha. (4-Boc amino butylene)glycine
A solution of 0.025 mole of N.sup..alpha. (Bzl)(4-Boc amino
butylene)glycine benzyl ester (Compound 70) in 60 mL MeOH was
hydrogenated(Procedure 8). Yield 89.5% of white solid. mp 176-8.degree. C.
TLC (Method A) Rf=0.23 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
COMPOUND 75
N.sup..alpha. (6-Boc amino hexylene)glycine
A solution of 0.025 mole of N.sup..alpha. (Bzl)(6-Boc amino
hexylene)glycine benzyl ester (Compound 71) in 60 mL MeOH was hydrogenated
(Procedure 8). Yield 80% of white solid. mp 172-4.degree. C.
TLC (Method A) Rf=0.26 (one spot); NMR (CDCl.sub.3) in agreement with the
title compound.
EXAMPLE 22
COMPOUND 76
N.sup..alpha. (Fmoc)(2-Boc amino ethylene)glycine
A solution of 0.02 mole of N.sup..alpha. (2-Boc amino ethylene)glycine
(Compound 72) in H.sub.2 O:Et.sub.3 N was reacted with FmocOSu (Procedure
9). Yield 80% of white solid. mp 130-132.degree. C. TLC (Method D) Rf=0.5
(one spot)
NMR (CDCl.sub.3) in agreement with the title compound.
Elemental Analysis: % C % H % N
Found: 65.18 6.11 5.91
Calc: 65.43 6.40 6.63
EXAMPLE 23
COMPOUND 77
N.sup..alpha. (Fmoc)(3-Boc amino propylene)glycine
A solution of 0.02 mole of N.sup..alpha. (Fmoc)(3-Boc amino
propylene)glycine (Compound 73) in H.sub.2 O:Et.sub.3 N was reacted with
FmocOSu (Procedure 9). Yield 85% of white solid. mp 125.degree. C.
TLC (Method D) Rf=0.5-0.6 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
Elemental Analysis: % C % H % N
Found: 66.05 6.65 6.00
Calc: 66.06 6.65 6.16
EXAMPLE 24
COMPOUND 78
N.sup..alpha. (Fmoc)(4-Boc amino butylene)glycine
A solution of 0.02 mole of N.sup..alpha. (Fmoc)(4-Boc amino
butylene)glycine (Compound 74) in H.sub.2 O:Et.sub.3 N was reacted with
FmocOSu (Procedure 9). Yield 79.4% of white solid. mp 150-152.degree. C.
TLC (Method D) Rf=0.42-0.47 (one spot); NMR (CDCl.sub.3) in agreement with
the title compound.
Elemental Analysis: % C % H % N
Found: 66.35 6.84 5.77
Calc: 66.06 6.88 5.98
EXAMPLE 25
COMPOUND 79
N.sup..alpha. (Fmoc)(6-Boc amino hexylene)glycine
A solution of 0.02 mole of N.sup..alpha. (Fmoc)(6-Boc amino
hexylene)glycine (Compound 75) in H.sub.2 O:Et.sub.3 N was reacted with
FmocOSu (Procedure 9). Yield 81.5% of white solid. mp 78-80.degree. C. TLC
(Method D) Rf=0.7 (one spot)
NMR (CDCl.sub.3) in agreement with the title compound.
Elemental Analysis: % C % H % N
Found: 68.02 7.08 5.37
Calc: 67.72 7.31 5.64
SYNTHETIC EXAMPLES
Two series of octapeptide somatostatin analogs of the present invention
were synthesized, characterized, and tested for biological activity.
1) The first series of compounds corresponds to the general Formula (XIVb);
this series comprises compounds of the specific formula
H-(D)Phe-R.sup.6 -Phe-(D)Trp-Lys-Thr-R.sup.11 -Thr-NH.sub.2
wherein R.sup.6 and R.sup.11 are N.sup..alpha. .omega.-functionalized
alkylene amino acid building units.
2) The second series of compounds corresponds to the general Formula
(XVIc); this series comprises compounds of the specific formula
H-(D) Phe-R.sup.6 -Phe-(D)Trp-Lys-R.sup.10 -Thr-NH.sub.2
wherein R.sup.6 and R.sup.10 are N.sup..alpha. .omega.-functionalized
alkylene amino acid building units.
The structures of these novel synthetic peptide analogs into which
N.sup..alpha. .omega.-functionalized amino acid building units were
incorporated, are summarized in Tables 7 and 8. In both series, the
building units used were glycine building units in which the bridging
groups, attached via the alpha nitrogens to the peptide backbone, were
varied.
For the sake of simplicity, these two series are referred to herein as the
SST Gly.sup.6,Gly.sup.11 and SST Gly.sup.6,Gly.sup.10 series,
respectively.
In each series, the position of the cyclization points was constant, while
the length and direction of the bridge was varied. Thus, C2,N2 refers to a
bridge consisting of an amide bond in which the carbonyl group is closer
to the amino end of the peptide and which contains two methylene groups
between the bridge amide and each of the backbone nitrogens involved in
the bridge.
Peptide assembly was carried out either manually or with an automatic
peptide synthesizer (Applied Biosystems Model 433A). Following peptide
assembly, de-protection of bridging groups that form the cyclization arms
was carried out with Pd(PPh.sub.3).sub.4 (palladium tetrakis triphenyl
phosphine) in the case of Allyl/Alloc protecting groups or with TFA in the
case of tBu/Boc protecting groups. For obtaining the linear (non-cyclized)
analog, the peptides were cleaved from the resin at this stage.
Cyclization of the peptides was carried out with PyBOP. Cleavage of the
peptides from the polymeric support was carried out with suitable reagents
depending on the type of resin used, e.g., with TFA for Rink amide type
resins and with HF for mBHA (para-methyl benzhydryl amine) type resins.
The crude products were characterized by analytical HPLC. The peptides
were purified by preparative reversed phase HPLC. The purified products
where characterized by analytical HPLC, mass spectroscopy, and amino acid
analysis.
TABLE 7
SST Gly.sup.6, Gly.sup.11
Example Bridging Compound Crude
No. Groups Number Method Yield
26 C1,N2 Cyclic DE-3-32-4 1 NA**
27 C1,N2 Linear DE-3-32-2 1 NA
28 C1,N3 Cyclic PTR 3004 2 79 mg
29 C1,N3 Linear PTR 3005 2 34 mg
30 C2,N2 Cyclic PTR 3002 1 NA
31 C2,N2 Linear PTR 3001 1 NA
32 C2,N3 Cyclic PTR 3007 2 40 mg
33 C2,N3 Linear PTR 3008 2 40 mg
34 N2,C2 Cyclic YD-9-1661-1 2 NA
35 N2,C2 Linear YD-9-168-1 2 NA
36 N3,C2 Cyclic PTR 3010 2 100 mg
37 N3,C2 Linear PTR 3011 2 NA
38 Linear* PTR 3003 3 96 mg
*Linear refers to the identical sequence with Gly residues in place of
R.sup.6 and R.sup.11.
**NA denotes not available.
Table 7 methods:
1) Manual synthesis on mBHA resin. HF cleavage.
2) Manual synthesis on Rapp tentagel resin. TFA cleavage.
3) Rink amide resin; assembly in automated peptide synthesizer, 0.1 mmol
scale.
TABLE 8
SST Gly.sup.6, Gly.sup.10
Example Bridging Compound Crude
No. Groups Number Method Yield
39 C1,N2 Cyclic YD-9-171-3 1 20 mg
40 C1,N2 Linear YD-9-171-2 1 10 mg
41 C1,N3 Cyclic YD-9-175-3 1 44.9 mg
42 C1,N3 Linear YD-9-175-2 1 25.4 mg
43 C2,N2 Cyclic PTR 3019 1 40 mg
44 C2,N2 Linear PTR 3020 1 26 mg
45 C2,N3 Cyclic YD-5-28-3 3 101.5 mg
46 C2,N3 Linear YD-5-28-2 3 48.3 mg
47 N2,C2 Cyclic PTR 3016 2 60 mg
48 N2,C2 Linear PTR 3017 2 40 mg
49 N3,C2 Cyclic YS-8-153-1 2 93 mg
50 N3,C2 Linear YS-8-152-1 2 54 mg
51 *Linear PTR 3021 1 100 mg
**Acetylated
Des-D-Phe.sup.5
52 N3,C2 Cyclic PTR 3013 67 mg
53 N3,C2 Linear PTR 3014 48 mg
*Linear refers to the identical sequence with Gly residues in place of
R.sup.6 and R.sup.10.
**Acetylated Des-D-Phe.sup.5 refers to the same sequence in which the N
terminal D-Phe.sup.5 is absent and the N-terminus is acetylated.
Table 8 methods:
1) Assembly in automated peptide synthesizer; 0.1 mmol scale. (HBTU).
2) Manual synthesis; PyBrop.
3) Assembly in automated peptide synthesizer, 0.25 mmol scale. (HBTU).
Synthesis of SST Gly.sup.6,Gly.sup.10 N3,C2:
Five grams of Rink amide resin (NOVA) (0.49 mmol/g), were swelled in
N-methylpyrrolidone (NMP) in a reaction vessel equipped with a sintered
glass bottom and placed on a shaker. The Fmoc protecting group was removed
from the resin by reaction with 20% piperidine in NMP (2 times 10 minutes,
25 ml each). Fmoc removal was monitored by ultraviolet absorption
measurement at 290 nm. A coupling cycle was carried out with
Fmoc-Thr(OtBu)-OH (3 equivalents) PyBrop (3 equivalents) DIEA (6
equivalents) in NMP (20 ml) for 2 hours at room temperature. Reaction
completion was monitored by the qualitative ninhydrin test (Kaiser test).
Following coupling, the peptide-resin was washed with NMP (7 times with 25
ml NMP, 2 minutes each). Capping was carried out by reaction of the
peptide-resin with acetic anhydride (capping mixture: HOBt 400 mg, NMP 20
ml, acetic anhydride 10 ml, DIEA 4.4 ml) for 0.5 hours at room
temperature. After capping, NMP washes were carried out as above (7 times,
2 minutes each). Fmoc removal was carried out as above. Fmoc-Phe-OH was
coupled in the same manner, and the Fmoc group removed, as above. The
peptide resin was reacted with Fmoc-Gly-C2 (Allyl) building unit: coupling
conditions were as above. Fmoc removal was carried out as above.
Fmoc-Lys(Boc)-OH was coupled to the peptide resin by reaction with HATU (3
equivalents) and DIEA (6 equivalents) at room temperature overnight and
then at 50.degree. C. for one hour. Additional DIEA was added during
reaction to maintain a basic medium (as determined by pH paper to be about
9). This coupling was repeated. Coupling completion was monitored by the
Fmoc test (a sample of the peptide resin was taken and weighed, the Fmoc
was removed as above, and the ultraviolet absorption was measured).
Fmoc-D-Trp-OH was coupled to the peptide resin with PyBrop, as described
above. Following Fmoc removal, Fmoc-Phe-OH was coupled in the same way.
Synthesis was continued with one-fifth of the peptide resin.
Following Fmoc removal, the second building unit was introduced:
Fmoc-Gly-N3(Alloc)-OH by reaction with PYBrop, as described above. Capping
was carried out as described above. Following Fmoc removal, the
peptide-resin was divided into two equal portions. Synthesis was continued
with one of these portions. Boc-D-Phe-OH was coupled by reaction with
HATU, as described above for Fmoc-Lys(Boc)-OH. Capping was carried out as
above.
The Allyl and Alloc protecting groups were removed by reaction with
Pd(PPh.sub.3).sub.4 and acetic acid 5%, morpholine 2.5% in chloroform,
under argon, for 2 hours at room temperature. The peptide resin was washed
with NMP as above. Two-thirds of the resin were taken for cyclization.
Cyclization was carried out with PyBOP 3 equivalents, DIEA 6 equivalents,
in NMP, at room temperature overnight. The peptide resin was washed and
dried. The peptide was cleaved from the resin by reaction with TFA 81.5%,
phenol 5%, water 5%, EDT 2.5%, TIS (tri-isopropyl-silane) 1%, and 5%
methylene chloride, at 0.degree. C. for 15 minutes and 2 hours at room
temperature under argon. The mixture was filtered into cold ether (30 ml,
0.degree. C.) and the resin was washed with a small volume of TFA. The
filtrate was placed in a rotary evaporator and all the volatile components
were removed. An oily product was obtained. It was triturated with ether
and the ether decanted, three times. A white powder was obtained. This
crude product was dried. The weight of the crude product was 93 mg.
PHYSIOLOGICAL EXAMPLES
EXAMPLE 54
BRADYKININ ANTAGONIST ASSAY (Displacement of (.sup.3 H)dopamine release
from PC 12 cells)
Novel backbone cyclized peptide analogs of the present invention were
assayed in vitro for bradykinin antagonist activity by protection of
(.sup.3 H)dopamine release from PC 12 cells that express bradykinin
receptors. PC12 cells were grown in Dulbecco Modified Eagle's medium with
high glucose, supplemented with 10% horse serum, 5% fetal calf serum, 130
units/ml penicillin and 0.1 mg/ml streptomycin. For experiments, cells
were removed from the medium using 1 mmole EDTA and replated on collagen
coated-12-well plates and assayed 24 hr later. Release of (.sup.3
H)dopamine was determined as follows: cells were incubated for 1.5 hr at
37.degree. C. with 0.5 ml of growth medium and 0.85 ml (.sup.3 H)DA (41
Ci/mmole) and 10 mg/ml pargyline followed by extensive washing with medium
(3.times.1 ml) and release buffer consisting of (mM): 130 NaCl; 5 KCl; 25
NaHCO.sub.3 ; 1 NaH.sub.2 PO.sub.4 ; 10 glucose and 1.8 CaCl.sub.2. In a
typical experiment, cells were incubated with 0.5 ml buffer for 5
consecutive incubation periods of 3 min each at 37.degree. C. Spontaneous
(.sup.3 H)DA release was measured by collecting the medium released by the
cells successively for the first 3 min period. Antagonists were added to
the cells 3 min prior to stimulation (at the second period), and
stimulation of (.sup.3 H)DA release by 100 nmole of bradykinin are
monitored during the 3 period by 60 mmole KCl. The remaining of the
(.sup.3 H)DA was extracted from the cells by over night incubation with
0.5 ml 0.1 N HCl. (.sup.3 H)DA release during each 3 min period was
expressed as a % of the total (.sup.3 H)DA content of the cells. Net
evoked release was calculated from (.sup.3 H)DA release during stimulation
period after subtracting basal (.sup.3 H)DA release in the preceding
baseline period if not indicated otherwise.
At 10.sup.-6 M, Example 1 showed 30% inhibition of BK activity, Example 4
showed 17% inhibition of BK activity. Note, the noncyclized (control)
peptide of example 2 showed 0% inhibition of BK activity.
EXAMPLE 55
BRADYKININ ANTAGONIST ASSAY (Guinea-pig assay)
The ileum of the guinea-pig was selected as the preparation for the
bioassay. This tissue contains predominantly BK.sub.2 receptors. The
preparation consists of the longitudinal muscle layer with the adhering
mesenteric plexus. The isolated preparation was kept in Krebs solution and
contractions were measured with an isometric force transducer. The
guinea-pig ileum is highly sensitive to BK, with EC.sub.50 at
2.times.10.sup.-8 M. At least two control responses to BK
(2.times.10.sup.-8 M) were measured previous to measuring the responses of
backbone cyclized peptides of the present invention. Atropine (1 .mu.M)
was always present.
At 10.sup.-6 M, Example 1 showed 24% inhibition of BK activity, Example 3
showed 10% and Example 4 showed 17% inhibition of BK activity. Note, the
noncyclized (control) peptide of example 2 showed 0% inhibition of BK
activity.
EXAMPLE 56
SOMATOSTATIN ASSAY (Receptor based screening)
Initial screening is conducted using .sup.125 I-labeled SST analogs and
pituitary membrane preparations or cell lines. The binding assay is
described in Tran, V. T., Beal, M. F. and Martin, J. B. Science,
228:294-495, 1985, which is incorporated herein by reference in its
entirety and is optimized with regards to membrane concentration,
temperature and time. The assay is sensitive (nM range) and robust.
Selectivity will be based on the recent cloning of the five human SST
receptors. The ability to screen the compounds with regard to binding and
biological activity in mammalian cells should facilitate the development
of subtype-selective analogs. These compounds are useful in the treatment
of specific endocrine disorders and therefore should be devoid of unwanted
side effects.
EXAMPLE 57
SOMATOSTATIN (SST) ASSAY (In vivo assays)
The biological effects of SST on growth hormone, insulin and glucagon
release is conducted by measuring the levels of these hormones using
commercially available RIA test kits. Pharmacological effects of SST in
patients with neuroendocrine tumors of the gut will require determination
of 5-hydroxyindole acetic acid (for carcinoid) and VIP (for VIPoma). In
vivo visualization of SST receptor-positive tumors is performed as
described by Lambert et al., New England J. Med., 323:1246-1249 1990,
following i.v. administration of radio-iodinated SST analogs.
EXAMPLE 58
Receptor Binding Specificity of Cyclic Peptide Analogs
Binding of representative peptides of Examples 39-54 to different
somatostatin receptors was measured in vitro, in Chinese Hamster Ovary
(CHO) cells expressing the various receptors. An example of the
selectivity obtained with the cyclic peptides is presented in Table 9. The
values presented are percent inhibition of radioactive iodinated
somatostatin (SRIF-14) binding.
TABLE 9
Binding of peptide analogs to somatostatin receptor subtypes
Somatostatin Receptor (SSTR) Subtype
SSTR 2B SSTR 5
Compound Conc. (M)
Description 10.sup.-6 10.sup.-7 10.sup.-8 10.sup.-6
10.sup.-7 10.sup.-8
Compound
Number
PTR 3003 Linear 16 3 0 55 20 0
PTR 3004 Cyclic C1,N3 0 0 0 14 0 0
PTR 3005 Linear C1,N3 0 0 0 9 0 0
PTR 3007 Cyclic C2,N3 0 0 0 19 9 0
PTR 3008 Linear C2,N3 0 0 0 15 6 0
PTR 3010 Cyclic N3,C2 0 0 0 63 26 9
PTR 3011 Cyclic N3,C2 0 0 0 27 66 27
Control
Peptides
BIM 3503 Pos. Control 81 33 16 92 66 27
PTR 4003 Neg. Control 0 0 0 0 0 0
EXAMPLE 59
Resistance to Biodegradation of SST Analogs
The in vitro biostability of a SST cyclic peptide analog, PTR 3002, was
measured in human serum, and was compared to the same sequence in a
non-cyclic peptide analog (PTR 3001), to octreotide (Sandostatin), and to
native somatostatin (SRIF). The results are shown in FIG. 1. In this
assay, the cyclic peptide in accordance with the present invention is as
stable as octreotide, is more stable than the corresponding non-cyclic
structure, and is much more stable than SRIF. The assay was based on HPLC
determination of peptide degradation as a function of time at 37.degree.
C.
EXAMPLE 60
Inhibition of Growth Hormone Release by SST Analogs
In vivo determination of the pharmacodynamic properties of cyclic peptide
analogs was carried out. Inhibition of Growth Hormone (GH) release as a
result of peptide administration was measured. Measurements were carried
out in Sprague-Dawley male rats: peptide analog activity was compared in
this study to SRIF or to octreotide (Sandostatin). Each group consisted of
4 rats. Time course profiles for GH release under constant experimental
conditions were measured.
Methods
Adult male Sprague-Dawley rats, specific pathogen free (SPF), weighing
200-350 g, were maintained on a constant light-dark cycle (light from 8:00
to 20:00 h), temperature (21.+-.3.degree. C.), and relative humidity
(55.+-.10%). Laboratory chow and tap water were available ad libitum. On
the day of the experiment, rats were anesthetized with pentobarbitone (50
mg/kg). Rats anesthetized with pentobarbitone exhibit low somatostatin
levels in portal blood vessels. (Plotsky, P. M., Science, 230, 461-463,
1985). A single blood sample (0.6 ml) was taken from the exposed
cannulated jugular vein for the determination of the basal GH levels (-15
min). Immediately thereafter the appropriate peptide pretreatment was
administered. The animals received 10 .mu.g/kg of either native
somatostatin (SRIF) or the synthetic analog octreotide (Sandostatin), or
the cyclic peptide analog. A saline solution (0.9% NaCl) was administered
as a control. All peptides were administered subcutaneously in a final
volume of 0.2 ml. Further sampling was carried out at 15, 30, 60, and 90
minutes after peptide administration. Immediately after the collection of
each blood sample, an appropriate volume (0.6 ml) of saline was
administered intravenously. Blood samples were collected into tubes
containing heparin (15 unites per ml of blood) and centrifuged
immediately. Plasma was separated and kept frozen at -20.degree. C. until
assayed.
Rat growth hormone (rGH) [.sup.125 I] levels were determined by appropriate
radioimmunoassay kit (Amersham). The standard in this kit has been
calibrated against a reference standard preparation (NIH-RP2) obtained
from the National Institute of Diabetes and Digestive and Kidney Diseases.
All samples were measured in duplicate.
EXAMPLE 61
Lack of Toxicity of Cyclized Peptide Analogs
PTR 3007 at a dose of 1.5 mg/kg was well tolerated after single
intraperitoneal application. PTR 3013 was not toxic to the rats even with
doses of 4 mg/kg. These two doses are several orders of magnitude higher
than those needed to elicit the desired endocrine effect. The peptides
dissolved in saline produced no untoward side effects on the central
nervous system, cardiovascular system, body temperature, nor on the
periphery of the animals. Rats were observed for 4 hours post
administration of the peptides. PTR 3007 and 3013 produced no respiratory
disturbances, did not result in the appearance of stereotyped behavior, or
produce any changes in muscle tone. After 3 hours, postmortem examination
did not detect any abnormality in the liver, kidneys, arteries and veins,
gastrointestinal tract, lungs, genital system, nor the spleen.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 2
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: not provided
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
1 5 10
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: not provided
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
Arg Pro Pro Gly Phe Ser Pro Phe Arg
1 5
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